Human monoclonal antibodies that bind CXCR4

ABSTRACT

The present disclosure provides isolated monoclonal antibodies that specifically bind to CXCR4 with high affinity, particularly human monoclonal antibodies. Nucleic acid molecules encoding the antibodies of this disclosure, expression vectors, host cells and methods for expressing the antibodies of this disclosure are also provided. Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of this disclosure are also provided. This disclosure also provides methods for detecting CXCR4, as well as methods for treating various cancers, inflammatory disorders and HIV infection using an anti-CXCR4 antibody of this disclosure.

BACKGROUND

Chemokines are a family of about 50 small proteins that modulate celltrafficking and angiogenesis and also play a significant role in thetumor microenvironment (Vicari, A. P. and Caux, C. (2002) CytokineGrowth Factor Rev. 13:143-154). Depending on their structure, chemokinesare classified as C-C chemokines (containing a cysteine-cysteine motif)or C-X-C chemokines (containing a cysteine-X-cysteine motif). Receptorsthat bind such chemokines thus are classified as members of the CCRfamily or CXCR family, respectively. One member of the CXCR family isCXCR4, a seven transmembrane G-protein coupled receptor that ispredominantly expressed on lymphocytes and that activates chemotaxis.CXCR4 binds the chemokine CXCL12 (SDF-1).

CXCR4 plays a role in embryogenesis, homeostasis and inflammation.Studies with mice engineered to be deficient in CXCR4 or SDF-1 implicatethe CXCR4/SDF-1 pathway in organ vascularization, as well as in theimmune and hematopoietic systems (Tachibana, K. et al. (1998) Nature393:591-594). Moreover, CXCR4 has been shown to function as a coreceptorfor T lymphotrophic HIV-1 isolates (Feng, Y. et al. (1996) Science272:872-877). CXCR4 also has been shown to be expressed on a widevariety of cancer cell types. Additionally, the CXCR4/SDF-1 pathway hasbeen shown to be involved in stimulating the metastatic process in manydifferent neoplasms (Murphy, P. M. (2001) N. Engl. Med. 345:833-835).For example, CXCR4 and SDF-1 have been shown to mediate organ-specificmetastasis by creating a chemotactic gradient between the primary tumorsite and the metastatic site (Muller, A. et al. (2001) Nature 410:50-56;Murakami, T. et al. (2002) Cancer Res. 62:7328-7334; Hanahan, D. et al.(2003) Cancer Res. 63:3005-3008).

SUMMARY

The present disclosure provides isolated monoclonal antibodies, inparticular human monoclonal antibodies, that bind to human CXCR4 andthat exhibit numerous desirable properties. These properties include theability to bind to native human CXCR4 expressed on a cell surface, theability to inhibit SDF-1 binding to human CXCR4, the ability to inhibitSDF-1-induced calcium flux in cells expressing CXCR4, the ability toinhibit SDF-1-induced migration of cells expressing CXCR4, the abilityto inhibit capillary tube formation by human umbilical vein endothelialcells (HuVECs), the ability to induce apoptosis in cells expressingCXCR4, the ability to inhibit tumor cell proliferation in vitro, theability to inhibit tumor cell proliferation in vivo, the ability toinhibit metastases of CXCR4⁺ tumor cells and/or the ability to increasesurvival time of a CXCR4⁺ tumor-bearing subject.

In one aspect, the instant disclosure pertains to an isolated humanmonoclonal antibody, or an antigen-binding portion thereof, wherein theantibody binds to native human CXCR4 expressed on a cell surface. In oneembodiment, the antibody also inhibits binding of SDF-1 to human CXCR4,preferably with an EC₅₀ for inhibition of 50 nM or less, or 30 nM orless, or 15 nM or less, or 10 nM or less, or 5 nM or less, or 3 nM orless (e.g., an EC₅₀ for inhibition of 28.60 nM or less, or 12.51 nM orless, or 2.256 nM or less). In another embodiment, the antibody binds tonative human CXCR4 expressed on a cell surface but does not inhibitbinding of SDF-1 to human CXCR4. In yet other embodiments, the antibodyalso inhibits SDF-1-induced calcium flux in cells expressing humanCXCR4, preferably with an EC₅₀ for inhibition of 3 nM or less, or 2 nMor less, or 1 nM or less, or 0.9 nM or less, or 0.8 nM or less, or 0.7nM or less, or 0.6 nM or less, or 0.5 nM or less, or 0.4 nM or less(e.g., 0.9046 nM or less, 0.5684 or less, or 0.3219 nM or less). In yetother embodiments, the antibody also inhibits SDF-1-induced migration ofcells expressing human CXCR4, preferably with an EC₅₀ for inhibition of50 nM or less, or 30 nM or less, or 20 nM or less, or 15 nM or less(e.g., 18.99 nM or less, or 12.44 or less). In still other embodiments,the antibody also inhibits capillary tube formation by HuVECs, inducesapoptosis of cells expressing CXCR4, inhibits tumor cell proliferationin vitro, inhibits tumor cell proliferation or induces tumor cellapoptosis in vivo, inhibits metastases of CXCR4⁺ tumor cells and/orincreases survival time of a CXCR4⁺ tumor-bearing subject.

Preferably, the antibody binds to human CXCR4 with high affinity, suchas with a K_(D) of 1×10⁻⁷ M or less or with a K_(D) of 5×10⁻⁸ M or less.Preferably, the antibodies of this disclosure are full-length antibodies(i.e., comprising variable and constant regions). Furthermore, theantibodies of this disclosure preferably are raised against full-lengthhuman CXCR-4 expressed in its native conformation on a host cell or inan artificial membrane.

In a preferred aspect, this disclosure pertains to an isolated humanmonoclonal antibody, or an antigen-binding portion thereof, wherein theantibody:

-   -   (a) binds to native human CXCR4 expressed on a cell surface;    -   (b) inhibits binding of SDF-1 to human CXCR4;    -   (c) inhibits SDF-1-induced calcium flux in cells expressing        human CXCR4;    -   (d) inhibits SDF-1-induced migration of cells expressing human        CXCR4; and    -   (e) inhibits capillary tube formation by human umbilical vein        endothelial cells.        Even more preferably, the antibody also induces apoptosis of        cells expressing human CXCR4 and/or inhibits growth of CXCR4⁺        tumor cells and/or induces tumor cell apoptosis in vivo.

In another aspect, this disclosure pertains to an isolated humanmonoclonal antibody, or antigen binding portion thereof, wherein theantibody cross-competes for binding to CXCR4 with a reference antibody,wherein the reference antibody comprises:

-   -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 25 and a light chain variable region        comprising the amino acid sequence of SEQ ID NO: 29; or    -   (b) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 26 and a light chain variable region        comprising the amino acid sequence of SEQ ID NO: 30; or    -   (c) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 27 and a light chain variable region        comprising the amino acid sequence of SEQ ID NO: 31; or    -   (d) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 28 and a light chain variable region        comprising the amino acid sequence of SEQ ID NO: 32.

In certain embodiments, this disclosure provides an isolated monoclonalantibody, or an antigen-binding portion thereof, comprising a heavychain variable region that is the product of or derived from a humanV_(H) 3-48 gene, wherein the antibody specifically binds human CXCR4. Inother embodiments, this disclosure provides an isolated monoclonalantibody, or an antigen-binding portion thereof, comprising a lightchain variable region that is the product of or derived from a humanV_(K) L15 gene, wherein the antibody specifically binds human CXCR4. Inyet other embodiments, this disclosure provides an isolated monoclonalantibody, or an antigen-binding portion thereof, comprising a heavychain variable region that is the product of or derived from a humanV_(H) 3-48 gene and a light chain variable region that is the product ofor derived from a human V_(K) L15 gene, wherein the antibodyspecifically binds human CXCR4.

In another aspect, this disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, comprising:

-   -   a heavy chain variable region that comprises CDR1, CDR2, and        CDR3 sequences; and a light chain variable region that comprises        CDR1, CDR2, and CDR3 sequences, wherein:    -   (a) the heavy chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequences of SEQ ID NOs: 9-12, and conservative        modifications thereof;    -   (b) the light chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequence of SEQ ID NOs: 21-24, and conservative        modifications thereof; and    -   (c) the antibody binds to native human CXCR4 expressed on a cell        surface.        In preferred embodiments, this antibody also has one or more of        the following characteristics: inhibits binding of SDF-1 to        CXCR4, inhibits SDF-1-induced calcium flux in cells expressing        CXCR4, inhibits SDF-1-induced migration of cells expressing        CXCR-4; inhibits capillary tube formation by HuVECs; induces        apoptosis in cells expressing CXCR4 (in vitro and/or in vivo),        inhibits growth of CXCR4⁺ tumor cells in vitro and/or in vivo,        and/or inhibits metatases of CXCR4⁺ tumor cells.

Preferably, the heavy chain variable region CDR2 sequence comprises anamino acid sequence selected from the group consisting of amino acidsequences of SEQ ID NOs: 5-8, and conservative modifications thereof,and the light chain variable region CDR2 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequencesof SEQ ID NOs: 17-20, and conservative modifications thereof.Preferably, the heavy chain variable region CDR1 sequence comprises anamino acid sequence selected from the group consisting of amino acidsequences of SEQ ID NOs: 1-4, and conservative modifications thereof;and the light chain variable region CDR1 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequencesof SEQ ID NOs: 13-16, and conservative modifications thereof.

A preferred combination comprises:

-   -   (a) heavy chain variable region CDR1 comprising SEQ ID NO: 1;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 5;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 9;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO: 13;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO: 17;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO: 21.

Another preferred combination comprises:

-   -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 2;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 6;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 10;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO: 14;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO: 18;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO: 22.

Another preferred combination comprises:

-   -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 3;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 7;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 11;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO: 15;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO: 19;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO: 23.

Another preferred combination comprises:

-   -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 4;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 8;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 12;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO: 16;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO: 20;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO: 24.

Other preferred antibodies of this disclosure, or antigen bindingportions thereof, comprise: (a) a heavy chain variable region comprisingan amino acid sequence selected from the group consisting of SEQ ID NOs:25-28 and 41-44; and (b) a light chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:29-32 and 45-48; wherein the antibody specifically binds CXCR4.

A preferred combination comprises: (a) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 25 or 41; and (b) alight chain variable region comprising the amino acid sequence of SEQ IDNO: 29 or 45.

Another preferred combination comprises: (a) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 26 or 42; and(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 30 or 46.

Another preferred combination comprises: (a) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 27 or 43; and(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 31 or 47.

Another preferred combination comprises: (a) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 28 or 44; and(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 32 or 48.

In another aspect of this disclosure, antibodies, or antigen-bindingportions thereof, are provided that compete for binding to CXCR4 withany of the aforementioned antibodies.

The antibodies of this disclosure can be, for example, full-lengthantibodies, for example of an IgG1 or IgG4 isotype. Alternatively, theantibodies can be antibody fragments, such as Fab, Fab′ or Fab′2fragments, or single chain antibodies.

This disclosure also provides an immunoconjugate comprising an antibodyof this disclosure, or antigen-binding portion thereof, linked to atherapeutic agent, such as a cytotoxin or a radioactive isotope. Thisdisclosure also provides a bispecific molecule comprising an antibody,or antigen-binding portion thereof, of this disclosure, linked to asecond functional moiety having a different binding specificity thansaid antibody, or antigen binding portion thereof.

Compositions comprising an antibody, or antigen-binding portion thereof,or immunoconjugate or bispecific molecule of this disclosure and apharmaceutically acceptable carrier are also provided.

Nucleic acid molecules encoding the antibodies, or antigen-bindingportions thereof, of this disclosure are also encompassed by thisdisclosure, as well as expression vectors comprising such nucleic acidsand host cells comprising such expression vectors. Methods for preparinganti-CXCR4 antibodies using the host cells comprising such expressionvectors are also provided and may include the steps of (i) expressingthe antibody in the host cell and (ii) isolating the antibody from thehost cell.

Another aspect of this disclosure pertains to methods of modulatingCXCR4 activity in a cell, wherein the cells are contacted with anantibody, or antigen-binding portion thereof, of this disclosure. Thecells can be contacted in vitro by culturing the cells with the antibodyor the cells can be contacted in vivo by administering the antibody to asubject. In a preferred embodiment, the cells are tumor cells expressingCXCR4 and the method results in inhibition of the growth of tumor cellsand/or inhibition of metastasis of the tumor cells. In anotherembodiment, the cells are T cells expressing CXCR4 and the methodresults in inhibition of entry of HIV into the cells. In yet anotherembodiment, the cells are lymphocytes in an inflammatory disorder andthe methods result in inhibition of inflammation. In yet anotherembodiment, the cells are involved in vascularization and the methodresults in modulation of angiogenesis.

In another aspect, this disclosure pertains to a method of stimulatingmobilization of CD34⁺ stem cells from bone marrow to peripheral blood ina subject, the method comprising administering to the subject anantibody, or antigen-binding portion thereof, of this disclosure suchthat mobilization of CD34⁺ stem cells from bone marrow to peripheralblood is stimulated. The method can further comprise collecting theCD34⁺ stem cells from the peripheral blood, such as for use inautologous stem cell transplantation.

Other features and advantages of the instant disclosure will be apparentfrom the following detailed description and examples, which should notbe construed as limiting. The contents of all references, Genbankentries, patents and published patent applications cited throughout thisapplication are expressly incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the nucleotide sequence (SEQ ID NO: 33) and amino acidsequence (SEQ ID NO: 25) of the heavy chain variable region of the F7human monoclonal antibody. The CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 5)and CDR3 (SEQ ID NO: 9) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 1B shows the nucleotide sequence (SEQ ID NO: 37) and amino acidsequence (SEQ ID NO: 29) of the light chain variable region of the F7human monoclonal antibody. The CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO:17) and CDR3 (SEQ ID NO: 21) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 2A shows the nucleotide sequence (SEQ ID NO: 34) and amino acidsequence (SEQ ID NO: 26) of the heavy chain variable region of the F9human monoclonal antibody. The CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 6)and CDR3 (SEQ ID NO: 10) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 2B shows the nucleotide sequence (SEQ ID NO: 38) and amino acidsequence (SEQ ID NO: 30) of the light chain variable region of the F9human monoclonal antibody. The CDR1 (SEQ ID NO: 14), CDR2 (SEQ ID NO:18) and CDR3 (SEQ ID NO: 22) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 3A shows the nucleotide sequence (SEQ ID NO: 35) and amino acidsequence (SEQ ID NO: 27) of the heavy chain variable region of the D1human monoclonal antibody. The CDR1 (SEQ ID NO: 3), CDR2 (SEQ ID NO: 7)and CDR3 (SEQ ID NO: 11) regions are delineated and the V, D and J germline derivations are indicated.

FIG. 3B shows the nucleotide sequence (SEQ ID NO: 39) and amino acidsequence (SEQ ID NO: 31) of the light chain variable region of the D1human monoclonal antibody. The CDR1 (SEQ ID NO: 15), CDR2 (SEQ ID NO:19) and CDR3 (SEQ ID NO: 23) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 4A shows the nucleotide sequence (SEQ ID NO: 36) and amino acidsequence (SEQ ID NO: 28) of the heavy chain variable region of the E2human monoclonal antibody. The CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 8)and CDR3 (SEQ ID NO: 12) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 4B shows the nucleotide sequence (SEQ ID NO: 40) and amino acidsequence (SEQ ID NO: 32) of the light chain variable region of the E2human monoclonal antibody. The CDR1 (SEQ ID NO: 16), CDR2 (SEQ ID NO:20) and CDR3 (SEQ ID NO: 24) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 5A shows the alignment of the amino acid sequence of the heavychain variable regions of F7 (SEQ ID NO: 25) and F7GL (SEQ ID NO: 41)with the human germline V_(H) 3-48 amino acid sequence (SEQ ID NO:49)(JH6b germline disclosed as SEQ ID NO: 52).

FIG. 5B shows the alignment of the amino acid sequence of the lightchain variable region of F7 (SEQ ID NO: 29) and F7GL (SEQ ID NO: 45)with the human germline V_(k) L15 amino acid sequence (SEQ ID NO:50)(JK1germline disclosed as SEQ ID NO: 53).

FIG. 6A shows the alignment of the amino acid sequence of the heavychain variable regions of F9 (SEQ ID NO: 26) and F9GL (SEQ ID NO: 42)with the human germline V_(H) 3-48 amino acid sequence (SEQ ID NO: 49)(JH6b germline disclosed as SEQ ID NO: 52).

FIG. 6B shows the alignment of the amino acid sequence of the lightchain variable region of F9 (SEQ ID NO: 30) and F9GL (SEQ ID NO: 46)with the human germline V_(k) L15 amino acid sequence (SEQ ID NO:50)(JK1 germline disclosed as SEQ ID NO: 53).

FIG. 7A shows the alignment of the amino acid sequence of the heavychain variable regions of D1 (SEQ ID NO: 27) and D1GL (SEQ ID NO: 43)with the human germline V_(H) 3-48 amino acid sequence (SEQ ID NO: 49)(JH6b germline disclosed as SEQ ID NO: 52).

FIG. 7B shows the alignment of the amino acid sequence of the lightchain variable region of D1 (SEQ ID NO: 31) and D1GL (SEQ ID NO: 47)with the human germline V_(k) L15 amino acid sequence (SEQ ID NO:50)(JK1 germline disclosed as SEQ ID NO: 53).

FIG. 8A shows the alignment of the amino acid sequence of the heavychain variable regions of E2 (SEQ ID NO: 28) and E2GL (SEQ ID NO: 44)with the human germline V_(H) 3-48 amino acid sequence (SEQ ID NO: 49)(JH6b germline disclosed as SEQ ID NO: 52).

FIG. 8B shows the alignment of the amino acid sequence of the lightchain variable region of E2 (SEQ ID NO: 32) and E2GL (SEQ ID NO: 48)with the human germline V_(k) L15 amino acid sequence (SEQ ID NO:50)(JK1 germline disclosed as SEQ ID NO: 53).

FIG. 9 is a graph showing the binding of anti-CXCR4 human antibodies F7,F9, D1 and E2 to CEM cells that express native human CXCR4 on the cellsurface.

FIG. 10 is a graph showing antibody competition for binding to CEM cellsbetween FITC-labeled anti-CXCR4 antibody F9 and a panel of unlabeledanti-CXCR4 human antibodies.

FIG. 11 is a graph showing inhibition of binding of ¹²⁵I-labeled SDF-1to CEM cells by anti-CXCR4 human antibodies F7, F9 and D1.

FIG. 12 is a graph showing inhibition of SDF-1-induced calcium flux inCEM cells by anti-CXCR4 human antibodies F7, F9 and D1.

FIG. 13 is a graph showing inhibition of SDF-1-induced migration of CEMcells by anti-CXCR4 human antibodies F7 and F9.

FIG. 14 is a graph showing inhibition of Ramos tumor cell proliferationin vitro by anti-CXCR4 human antibodies F7, F9 and E2.

FIGS. 15A-C are graphs showing inhibition of Ramos tumor cellproliferation in vivo in a subcutaneous tumor model by anti-CXCR4 humanantibodies F7 and F9. FIG. 15A shows the mean tumor volume growth curve;FIG. 15B shows the median tumor volume growth curve; FIG. 15C shows themedian % body weight change.

FIG. 16 is a graph showing % survival of mice treated with theanti-CXCR4 human antibody F9 in a Ramos systemic tumor cell model.

DETAILED DESCRIPTION OF THIS DISCLOSURE

The present disclosure relates to isolated monoclonal antibodies,particularly human monoclonal antibodies, which bind specifically tonative human CXCR4 expressed on a cell surface. In certain embodiments,the antibodies of this disclosure are derived from particular heavy andlight chain germline sequences and/or comprise particular structuralfeatures such as CDR regions comprising particular amino acid sequences.This disclosure provides isolated antibodies, methods of making suchantibodies, immunoconjugates and bispecific molecules comprising suchantibodies and pharmaceutical compositions containing the antibodies,immunoconjugates or bispecific molecules of this disclosure. Thisdisclosure also relates to methods of using the antibodies, such as todetect CXCR4, as well as to modulate CXCR4 activity in diseases ordisorders associated with expression of CXCR4 or involving theCXCR4/SDF-1 pathway, such as cancers, tumor metastasis, HIV infection,inflammation and angiogenesis. Accordingly, this disclosure alsoprovides methods of using the anti-CXCR4 antibodies of this disclosureto treat cancer, for example, to treat a cancer such as breast, ovarian,prostate, non-small cell lung, pancreatic, thyroid, melanoma,nasopharyngeal, renal cell, lymphoma, neuroblastoma, glioblastoma,rhabdomyosarcoma, colorectal, kidney, osteosarcoma, acute lymphoblasticleukemia or acute myeloid leukemia. Additionally, this disclosureprovides methods of using the anti-CXCR4 antibodies of this disclosureto inhibit tumor metastasis.

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “CXCR4” includes variants, isoforms, homologs, orthologs andparalogs. For example, antibodies specific for CXCR4 may, in certaincases, cross-react with CXCR4 from species other than human. In otherembodiments, the antibodies specific for human CXCR4 may be completelyspecific for human CXCR4 and may not exhibit species or other types ofcross-reactivity. The term “human CXCR4” refers to human sequence CXCR4,such as the complete amino acid sequence of human CXCR4 having Genbankaccession number P61073 (SEQ ID NO.:51). CXCR4 is also known in the artas, for example, LESTR, Fusin or CD184. The human CXCR4 sequence maydiffer from human CXCR4 of SEQ ID NO.:51 by having, for example,conserved mutations or mutations in non-conserved regions and the CXCR4has substantially the same biological function as the human CXCR4 of SEQID NO.:51. For example, a biological function of human CXCR4 is havingan epitope in the extracellular domain of CXCR4 that is specificallybound by an antibody of the instant disclosure or the biologicalfunction of human CXCR4 is chemokine binding or involvement in themetastatic process.

A particular human CXCR4 sequence will generally be at least 90%identical in amino acids sequence to human CXCR4 of SEQ ID NO.:51 andcontains amino acid residues that identify the amino acid sequence asbeing human when compared to CXCR4 amino acid sequences of other species(e.g., murine). In certain cases, a human CXCR4 may be at least 95%, oreven at least 96%, 97%, 98%, or 99% identical in amino acid sequence toCXCR4 of SEQ ID NO.:51. In certain embodiments, a human CXCR4 sequencewill display no more than 10 amino acid differences from the CXCR4 ofSEQ ID NO.:51. In certain embodiments, the human CXCR4 may display nomore than 5, or even no more than 4, 3, 2, or 1 amino acid differencefrom the CXCR4 of SEQ ID NO.:51. Percent identity can be determined asdescribed herein.

The term “SDF-1” refers to stromal cell-derived factor 1, which is aligand for CXCR4. The term “SDF-1” encompasses different isoforms ofSDF-1, such as SDF-1α and SDF-1β. The amino acid sequence of humanSDF-1α has Genbank accession number NP_(—)954637. The amino acidsequence of human SDF-1β has Genbank accession number NP_(—)000600.Human SDF-1 is also described in U.S. Pat. No. 5,756,084. SDF-1 is alsoknown as CXCL12. The amino acid sequence of human SDF-1 can differ fromthe SDF-1 of NP_(—)954637 or NP_(—)000600, as described herein forCXCR4.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. As used herein, the phrase “cell surface receptor”includes, for example, molecules and complexes of molecules capable ofreceiving a signal and the transmission of such a signal across theplasma membrane of a cell. An example of a “cell surface receptor” ofthe present disclosure is the CXCR4 receptor.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein as VH)and a heavy chain constant region. The heavy chain constant region iscomprised of three domains, C_(H)1, C_(H)2 and C_(H)3. Each light chainis comprised of a light chain variable region (abbreviated herein asV_(L)) and a light chain constant region. The light chain constantregion is comprised of one domain, C_(L). The V_(H) and V_(L) regionscan be further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., CXCR4). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H)1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fab′fragment, which is essentially an Fab with part of the hinge region(see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed. 1993); (iv) a Fdfragment consisting of the V_(H) and C_(H)1 domains; (v) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; (vii) an isolated complementaritydetermining region (CDR); and (viii) a nanobody, a heavy chain variableregion containing a single variable domain and two constant domains.Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et. al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies arealso intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds CXCR4 is substantially free of antibodies that specifically bindantigens other than CXCR4). An isolated antibody that specifically bindsCXCR4 may, however, have cross-reactivity to other antigens, such asCXCR4 molecules from other species. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies of this disclosure may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity, which have variable regions in which boththe framework and CDR regions are derived from human germlineimmunoglobulin sequences. In one embodiment, the human monoclonalantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic nonhuman animal, e.g., a transgenic mouse, having agenome comprising a human heavy chain transgene and a light chaintransgene fused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by the heavy chain constant region genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody.

The term “humanized antibody” is intended to refer to antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. Additional framework region modifications may be made withinthe human framework sequences.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

As used herein, an antibody that “specifically binds to human CXCR4” isintended to refer to an antibody that binds to human CXCR4 (and possiblyCXCR4 from one or more non-human species) but does not substantiallybind to non-CXCR4 proteins. In certain embodiments, an antibody of theinstant disclosure specifically binds to human CXCR4 of SEQ ID NO.:51 ora variant thereof. Preferably, the antibody binds to human CXCR4 with aK_(D) of 1×10⁻⁷ M or less, more preferably 5×10⁻⁸ M or less, morepreferably 3×10⁻⁸ M or less, more preferably 1×10⁻⁸ M or less, even morepreferably 5×10⁻⁹ M or less.

The term “does not substantially bind” to a protein or cells, as usedherein, means does not bind or does not bind with a high affinity to theprotein or cells, i.e. binds to the protein or cells with a K_(D) of1×10⁻⁶ M or more, more preferably 1×10⁻⁵ M or more, more preferably1×10⁻⁴ M or more, more preferably 1×10⁻³ M or more, even more preferably1×10⁻² M or more.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e., K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A preferred method for determining the K_(D) ofan antibody is by using surface plasmon resonance, preferably using abiosensor system such as a Biacore® system.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 1×10⁻⁷M or less, more preferably 5×10⁻⁸ Mor less, even more preferably 1×10⁻⁸M or less, even more preferably5×10⁻⁹ M or less and even more preferably 1×10⁻⁹ M or less for a targetantigen. However, “high affinity” binding can vary for other antibodyisotypes. For example, “high affinity” binding for an IgM isotype refersto an antibody having a K_(D) of 10⁻⁶ M or less, more preferably 10⁻⁷ Mor less, even more preferably 10⁻⁸ M or less.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows, chickens, amphibians, reptiles, etc.

Various aspects of this disclosure are described in further detail inthe following subsections.

Anti-CXCR4 Antibodies

The antibodies of this disclosure are characterized by particularfunctional features or properties of the antibodies. For example, theantibodies bind to native human CXCR4 expressed on a cell surface.Preferably, an antibody of this disclosure binds to CXCR4 with highaffinity, for example with a K_(D) of 1×10⁻⁷ M or less. The anti-CXCR4antibodies of this disclosure preferably exhibit one or more of thefollowing characteristics:

-   -   (a) binding to native human CXCR4 expressed on a cell surface;    -   (b) inhibiting binding of SDF-1 to CXCR4;    -   (c) inhibiting SDF-1-induced calcium flux in cells expressing        CXCR4;    -   (d) inhibiting SDF-1-induced migration of cells expressing        CXCR4;    -   (e) inhibiting capillary tube formation by human umbilical vein        endothelial cells;    -   (f) binding to human CXCR4 with a K_(D) of 1×10⁻⁷ M or less;    -   (g) inducing apoptosis in cells expressing CXCR4;    -   (h) inhibiting tumor cell proliferation in vitro;    -   (i) inhibiting tumor cell proliferation and/or inducing tumor        cell apoptosis in vivo;    -   (j) inhibiting metastases of CXCR4⁺ tumor cells; and/or    -   (k) increasing survival time of a CXCR4⁺ tumor-bearing subject.

In certain embodiments, an antibody of this disclosure binds to nativehuman CXCR4 on a cell surface but does not inhibit binding of SDF-1 toCXCR4 and does not inhibit SDF-1-induced calcium flux in cellsexpressing CXCR4 and does not inhibit SDF-1-induced migration of cellsexpressing CXCR4. In other embodiments, an antibody of this disclosurebinds to native human CXCR4 on a cell surface and does inhibit bindingof SDF-1 to CXCR4 and does inhibit SDF-1-induced calcium flux in cellsexpressing CXCR4 but does not inhibit SDF-1-induced migration of cellsexpressing CXCR4. In still other embodiments, an antibody of thisdisclosure binds to native human CXCR4 on a cell surface and doesinhibit binding of SDF-1 to CXCR4 and does inhibit SDF-1-induced calciumflux in cells expressing CXCR4 and does inhibit SDF-1-induced migrationof cells expressing CXCR4. In still other embodiments, an antibody ofthis disclosure binds to native human CXCR4 on a cell surface, doesinhibit binding of SDF-1 to CXCR4, does inhibit SDF-1-induced calciumflux in cells expressing CXCR4, does inhibit SDF-1-induced migration ofcells expressing CXCR4 and does inhibit capillary tube formation byHuVECs.

Preferably, an antibody of this disclosure binds to human CXCR4 with aK_(D) of 5×10⁻⁸ M or less, binds to human CXCR4 with a K_(D) of 2×10⁻⁸ Mor less, binds to human CXCR4 with a K_(D) of 5×10⁻⁹ M or less, binds tohuman CXCR4 with a K_(D) of 4×10⁻⁹ M or less, binds to human CXCR4 witha K_(D) of 3×10⁻⁹ M or less, or binds to human CXCR4 with a K_(D) of2×10⁻⁹ M or less.

Preferably, an antibody of the inhibits binding of SDF-1 to human CXCR4with an EC₅₀ for inhibition of 50 nM or less, more preferably 30 nM orless, or 15 nM or less, or 10 nM or less, or 5 nM or less, or 3 nM orless (e.g., an EC₅₀ for inhibition of 28.60 nM or less, or 12.51 nM orless, or 2.256 nM or less)

Preferably, an antibody of this disclosure inhibits SDF-1-inducedcalcium flux in cells expressing human CXCR4 with an EC₅₀ for inhibitionof 3 nM or less, more preferably 2 nM or less, or 1 nM or less, or 0.9nM or less, or 0.8 nM or less, or 0.7 nM or less, or 0.6 nM or less, or0.5 nM or less, or 0.4 nM or less (e.g., 0.9046 nM or less, 0.5684 orless, or 0.3219 nM or less).

Preferably, an antibody of this disclosure inhibits SDF-1-inducedmigration of cells expressing human CXCR4 with an EC₅₀ for inhibition of50 nM or less, more preferably 30 nM or less, or 20 nM or less, or 15 nMor less (e.g., 18.99 nM or less, or 12.44 or less).

Standard assays to evaluate the binding ability of the antibodies towardnative human CXCR4 expressed on a cell surface are known in the art,including for example, flow cytometry analysis using a cell line thatnaturally expresses native CXCR4 or that has been transfected to expressnative CXCR4. Suitable assays are described in detail in the Examples. Apreferred cell line that expresses native CXCR4 is the CEM T cell line.Suitable assays for evaluating inhibition of binding of SDF-1,inhibition of SDF-1 induced calcium flux, inhibition of SDF-1 inducedcell migration, inhibition of capillary tube formation by HuVECs,induction of apoptosis in cells expressing CXCR4 in vitro and/or invivo, inhibition of growth of CXCR4⁺ tumor cells in vitro and/or invivo, and/or inhibition of metastases of CXCR4⁺ tumor cells are alsodescribed in detail in the Examples. Binding affinity of the antibodiesalso can be determined by standard methods, such as by Scatchardanalysis.

Monoclonal Antibodies F7, F9, D1 and E2

Preferred antibodies of this disclosure are the human monoclonalantibodies F7, F9, D1 and E2, isolated and structurally characterized asdescribed in Examples 1 and 2. The V_(H) amino acid sequences of F7, F9,D1 and E2 are shown in SEQ ID NOs: 25, 26, 27 and 28, respectively. TheV_(L) amino acid sequences of F7, F9, D1 and E2 are shown in SEQ ID NOs:29, 30, 31 and 32, respectively. Additionally, alternative forms of F7,F9, D1 and E2, in which certain framework residues were substituted witha germline residue, were created and are referred to herein as F7GL,F9GL, D1GL and E2GL. The V_(H) amino acid sequences of F7GL, F9GL, D1GLand E2GL are shown in SEQ ID NOs: 41, 42, 43 and 44, respectively. TheV_(L) amino acid sequences of F7GL, F9GL, D1GL and E2GL are shown in SEQID NOs: 45, 46, 47 and 48, respectively.

Given that each of these antibodies can bind to CXCR4, the V_(H) andV_(L) sequences can be “mixed and matched” to create other anti-CXCR4binding molecules of this disclosure. CXCR4 binding of such “mixed andmatched” antibodies can be tested using the binding assays describedabove and in the Examples (e.g., flow cytometry with CEM cells).Preferably, when V_(H) and V_(L) chains are mixed and matched, a V_(H)sequence from a particular V_(H)/V_(L) pairing is replaced with astructurally similar V_(H) sequence. Likewise, preferably a V_(L)sequence from a particular V_(H)/V_(L) pairing is replaced with astructurally similar V_(L) sequence.

Accordingly, in one aspect, this disclosure provides an isolatedmonoclonal antibody, or antigen binding portion thereof comprising:

-   -   (a) a heavy chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 25-28        and 41-44; and    -   (b) a light chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 29-32        and 45-48;        wherein the antibody specifically binds CXCR4, preferably human        CXCR4.        Preferred heavy and light chain combinations include:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 25 or 41 and a light chain variable        region comprising the amino acid sequence of SEQ ID NO: 29 or        45; or    -   (b) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 26 or 42 and a light chain variable        region comprising the amino acid sequence of SEQ ID NO: 30 or        46; or    -   (c) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 27 or 43 and a light chain variable        region comprising the amino acid sequence of SEQ ID NO: 31 or        47; or    -   (d) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 28 or 44 and a light chain variable        region comprising the amino acid sequence of SEQ ID NO: 32 or        48.

In another aspect, this disclosure provides antibodies that comprise theheavy chain and light chain CDR1s, CDR2s and CDR3s of F7, F9, D1 or E2,or combinations thereof. The amino acid sequences of the V_(H) CDR1s ofF7, F9, D1 and E2 are shown in SEQ ID NOs: 1-4, respectively. The aminoacid sequences of the V_(H) CDR2s of F7, F9, D1 and E2 are shown in SEQID NOs: 5-8, respectively. The amino acid sequences of the V_(H) CDR3sof F7, F9, D1 and E2 are shown in SEQ ID NOs: 9-12, respectively. Theamino acid sequences of the V_(k) CDR1s of F7, F9, D1 and E2 are shownin SEQ ID NOs: 13-16, respectively. The amino acid sequences of theV_(k) CDR2s of F7, F9, D1 and E2 are shown in SEQ ID NOs: 17-20,respectively. The amino acid sequences of the V_(k) CDR3s of F7, F9, D1and E2 are shown in SEQ ID NOs: 21-24, respectively. The CDR regions aredelineated using the Kabat system (Kabat, E. A., et al. (1991) Sequencesof Proteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242).

Given that each of these antibodies can bind to CXCR4 and thatantigen-binding specificity is provided primarily by the CDR1, CDR2, andCDR3 regions, the V_(H) CDR1, CDR2, and CDR3 sequences and V_(k) CDR1,CDR2, and CDR3 sequences can be “mixed and matched” (i.e., CDRs fromdifferent antibodies can be mixed and match, although each antibody mustcontain a V_(H) CDR1, CDR2, and CDR3 and a V_(k) CDR1, CDR2, and CDR3)to create other anti-CXCR4 binding molecules of this disclosure. CXCR4binding of such “mixed and matched” antibodies can be tested using thebinding assays described above and in the Examples (e.g., ELISAs,Biacore® analysis). Preferably, when V_(H) CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular V_(H)sequence is replaced with a structurally similar CDR sequence(s).Likewise, when V_(k) CDR sequences are mixed and matched, the CDR1, CDR2and/or CDR3 sequence from a particular V_(k) sequence preferably isreplaced with a structurally similar CDR sequence(s). It will be readilyapparent to the ordinarily skilled artisan that novel V_(H) and V_(L)sequences can be created by substituting one or more V_(H) and/or V_(L)CDR region sequences with structurally similar sequences from the CDRsequences disclosed herein for monoclonal antibodies antibodies F7, F9,D1 and E2.

Accordingly, in another aspect, this disclosure provides an isolatedmonoclonal antibody, or antigen binding portion thereof comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-4;

(b) a heavy chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 5-8;

(c) a heavy chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 9-12;

(d) a light chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 13-16;

(e) a light chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 17-20; and

(f) a light chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 21-24;

wherein the antibody specifically binds CXCR4, preferably human CXCR4.

In a preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 1;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 5;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 9;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 13;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 17; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 21.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 2;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 6;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 10;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 14;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 18; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 22.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 3;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 7;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 11;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 15;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 19; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 23.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 4;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 8;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 12;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 16;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 20; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 24.

It is well known in the art that the CDR3 domain, independently from theCDR1 and/or CDR2 domain(s), alone can determine the binding specificityof an antibody for a cognate antigen and that multiple antibodies canpredictably be generated having the same binding specificity based on acommon CDR3 sequence. See, for example, Klimka et al., British J. ofCancer 83(2):252-260 (2000) (describing the production of a humanizedanti-CD30 antibody using only the heavy chain variable domain CDR3 ofmurine anti-CD30 antibody Ki-4); Beiboer et al., J. Mol. Biol.296:833-849 (2000) (describing recombinant epithelial glycoprotein-2(EGP-2) antibodies using only the heavy chain CDR3 sequence of theparental murine MOC-31 anti-EGP-2 antibody); Rader et al., Proc. Natl.Acad. Sci. U.S.A. 95:8910-8915 (1998) (describing a panel of humanizedanti-integrin α_(v)β₃ antibodies using a heavy and light chain variableCDR3 domain of a murine anti-integrin α_(v)β₃ antibody LM609 whereineach member antibody comprises a distinct sequence outside the CDR3domain and capable of binding the same epitope as the parent muringantibody with affinities as high or higher than the parent murineantibody); Barbas et al., J. Am. Chem. Soc. 116:2161-2162 (1994)(disclosing that the CDR3 domain provides the most significantcontribution to antigen binding); Barbas et al., Proc. Natl. Acad. Sci.U.S.A. 92:2529-2533 (1995) (describing the grafting of heavy chain CDR3sequences of three Fabs (SI-1, SI-40, and SI-32) against human placentalDNA onto the heavy chain of an anti-tetanus toxoid Fab thereby replacingthe existing heavy chain CDR3 and demonstrating that the CDR3 domainalone conferred binding specificity); Ditzel et al., J. Immunol.157:739-749 (1996) (describing grafting studies wherein transfer of onlythe heavy chain CDR3 of a parent polyspecific Fab LNA3 to a heavy chainof a monospecific IgG tetanus toxoid-binding Fab p313 antibody wassufficient to retain binding specificity of the parent Fab); Berezov etal., BIAjournal 8:Scientific Review 8 (2001) (describing peptidemimetics based on the CDR3 of an anti-HER2 monoclonal antibody; Igarashiet al., J. Biochem (Tokyo) 117:452-7 (1995) (describing a 12 amino acidsynthetic polypeptide corresponding to the CDR3 domain of ananti-phosphatidylserine antibody); Bourgeois et al., J. Virol 72:807-10(1998) (showing that a single peptide derived form the heavy chain CDR3domain of an anti-respiratory syncytial virus (RSV) antibody was capableof neutralizing the virus in vitro); Levi et al., Proc. Natl. Acad. Sci.U.S.A. 90:4374-8 (1993) (describing a peptide based on the heavy chainCDR3 domain of a murine anti-HIV antibody); Polymenis and Stoller, J.Immunol. 152:5218-5329 (1994) (describing enabling binding of an scFv bygrafting the heavy chain CDR3 region of a Z-DNA-binding antibody) and Xuand Davis, Immunity 13:37-45 (2000) (describing that diversity at theheavy chain CDR3 is sufficient to permit otherwise idential IgMmolecules to distinguish between a variety of hapten and proteinantigens). See also, U.S. Pat. Nos. 6,951,646; 6,914,128; 6,090,382;6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185,describing patented antibodies defined by a single CDR domain. Each ofthese references is hereby incorporated by reference in its entirety.

Accordingly, the present disclosure provides monoclonal antibodiescomprising one or more heavy and/or light chain CDR3 domains from anantibody derived from a human or non-human animal, wherein themonoclonal antibody is capable of specifically binding to CXCR4. Withincertain aspects, the present disclosure provides monoclonal antibodiescomprising one or more heavy and/or light chain CDR3 domain from anon-human antibody, such as a mouse or rat antibody, wherein themonoclonal antibody is capable of specifically binding to CXCR4. Withinsome embodiments, such inventive antibodies comprising one or more heavyand/or light chain CDR3 domain from a non-human antibody (a) are capableof competing for binding with; (b) retain the functionalcharacteristics; (c) bind to the same epitope; and/or (d) have a similarbinding affinity as the corresponding parental non-human antibody.

Within other aspects, the present disclosure provides monoclonalantibodies comprising one or more heavy and/or light chain CDR3 domainfrom a human antibody, such as, for example, a human antibody obtainedfrom a non-human animal, wherein the human antibody is capable ofspecifically binding to CXCR4. Within other aspects, the presentdisclosure provides monoclonal antibodies comprising one or more heavyand/or light chain CDR3 domain from a first human antibody, such as, forexample, a human antibody obtained from a non-human animal, wherein thefirst human antibody is capable of specifically binding to CXCR4 andwherein the CDR3 domain from the first human antibody replaces a CDR3domain in a human antibody that is lacking binding specificity for CXCR4to generate a second human antibody that is capable of specificallybinding to CXCR4. Within some embodiments, such inventive antibodiescomprising one or more heavy and/or light chain CDR3 domain from thefirst human antibody (a) are capable of competing for binding with; (b)retain the functional characteristics; (c) bind to the same epitope;and/or (d) have a similar binding affinity as the corresponding parentalfirst human antibody.

Antibodies Having Particular Germline Sequences

In certain embodiments, an antibody of this disclosure comprises a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene.

For example, in a preferred embodiment, this disclosure provides anisolated monoclonal antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region that is the product of orderived from a human V_(H) 3-48 gene, wherein the antibody specificallybinds CXCR4. In another preferred embodiment, this disclosure providesan isolated monoclonal antibody, or an antigen-binding portion thereof,comprising a light chain variable region that is the product of orderived from a human V_(K) L15 gene, wherein the antibody specificallybinds CXCR4. In yet another preferred embodiment, this disclosureprovides an isolated monoclonal antibody, or antigen-binding portionthereof, wherein the antibody comprises a heavy chain variable regionthat is the product of or derived from a human V_(H) 3-48 gene andcomprises a light chain variable region that is the product of orderived from a human V_(K) L15 gene, wherein the antibody specificallybinds to CXCR4, preferably human CXCR4. Such antibodies also may possessone or more of the functional characteristics described in detail above,such as binding to native CXCR4 expressed on a cell surface, inhibitionof SDF-1 binding to CXCR4, inhibition of SDF-1-induced calcium flux incells expressing CXCR4, inhibition of SDF-1-induced migration of cellsexpressing CXCR4, inhibition of capillary tube formation by HuVECs,induction of apoptosis in cells expressing CXCR4 in vitro and/or invivo, inhibition of growth of CXCR4⁺ tumor cells in vitro and/or invivo, and/or inhibition of metastases of CXCR4⁺ tumor cells.

Examples of antibodies having V_(H) and V_(K) of V_(H) 3-48 and V_(K)L15, respectively, are the F7, F9, D1 and E2 antibodies.

As used herein, a human antibody comprises heavy or light chain variableregions that is “the product of” or “derived from” a particular germlinesequence if the variable regions of the antibody are obtained from asystem that uses human germline immunoglobulin genes. Such systemsinclude immunizing a transgenic mouse carrying human immunoglobulingenes with the antigen of interest or screening a human immunoglobulingene library displayed on phage with the antigen of interest. A humanantibody that is “the product of” or “derived from” a human germlineimmunoglobulin sequence can be identified as such by comparing the aminoacid sequence of the human antibody to the amino acid sequences of humangermline immunoglobulins and selecting the human germline immunoglobulinsequence that is closest in sequence (i.e., greatest % identity) to thesequence of the human antibody. A human antibody that is “the productof” or “derived from” a particular human germline immunoglobulinsequence may contain amino acid differences as compared to the germlinesequence, due to, for example, naturally-occurring somatic mutations orintentional introduction of site-directed mutation. However, a selectedhuman antibody typically is at least 90% identical in amino acidssequence to an amino acid sequence encoded by a human germlineimmunoglobulin gene and contains amino acid residues that identify thehuman antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

Homologous Antibodies

In yet another embodiment, an antibody of this disclosure comprisesheavy and light chain variable regions comprising amino acid sequencesthat are homologous to the amino acid sequences of the preferredantibodies described herein, and wherein the antibodies retain thedesired functional properties of the anti-CXCR4 antibodies of thisdisclosure.

For example, this disclosure provides an isolated monoclonal antibody,or antigen binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein:

-   -   (a) the heavy chain variable region comprises an amino acid        sequence that is at least 80% homologous to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 25-28        and 41-44;    -   (b) the light chain variable region comprises an amino acid        sequence that is at least 80% homologous to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 29-32        and 45-48;    -   (c) the antibody binds to native human CXCR4 expressed on a cell        surface.

Additionally or alternatively, the antibody may possess one or more ofthe following functional properties: (i) binds to human CXCR4 with aK_(D) of 1×10⁷⁻⁷ M or less; (ii) inhibits SDF-1 binding to CXCR4; (iii)inhibits SDF-1-induced calcium flux in cells expressing CXCR4; (iv)inhibits SDF-1-induced migration of cells expressing CXCR4; (v) inhibitscapillary tube formation by HuVECs; (vi) induces apoptosis in cellsexpressing CXCR4 in vitro and/or in vivo; (vii) inhibits growth ofCXCR4⁺ tumor cells in vitro and/or in vivo; and/or (viii) inhibitsmetastases of CXCR4⁺ tumor cells.

In various embodiments, the antibody can be, for example, a humanantibody, a humanized antibody or a chimeric antibody.

In other embodiments, the V_(H) and/or V_(L) amino acid sequences may be85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences setforth above. An antibody having V_(H) and V_(L) regions having high(i.e., 80% or greater) homology to the V_(H) and V_(L) regions of thesequences set forth above, can be obtained by mutagenesis (e.g.,site-directed or PCR-mediated mutagenesis) of nucleic acid moleculesencoding SEQ ID NOs: 25-32 or 41-48, followed by testing of the encodedaltered antibody for retained function (i.e., the functions set forthabove) using the functional assays described herein.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentdisclosure can further be used as a “query sequence” to perform a searchagainst public databases to, for example, to identify related sequences.Such searches can be performed using theXBLAST program (version 2.0) ofAltschul, et al. (1990)J. Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody molecules of thisdisclosure. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are useful. See www.ncbi.nlm.nih.gov.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of this disclosure comprises a heavychain variable region comprising CDR1, CDR2 and CDR3 sequences and alight chain variable region comprising CDR1, CDR2 and CDR3 sequences,wherein one or more of these CDR sequences comprise specified amino acidsequences based on the preferred antibodies described herein (e.g., F7,F9, D1 or E2), or conservative modifications thereof, and wherein theantibodies retain the desired functional properties of the anti-CXCR4antibodies of this disclosure. It is understood in the art that certainconservative sequence modification can be made which do not removeantigen binding. See, for example, Brummell et al. (1993) Biochem32:1180-8 (describing mutational analysis in the CDR3 heavy chain domainof antibodies specific for Salmonella); de Wildt et al. (1997) Prot.Eng. 10:835-41 (describing mutation studies in anti-UA1 antibodies);Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870 (showing thatmutations in the middle of HCDR3 led to either abolished or diminishedaffinity); Hall et al. (1992) J. Immunol. 149:1605-12 (describing that asingle amino acid change in the CDR3 region abolished binding activity);Kelley and O'Connell (1993) Biochem. 32:6862-35 (describing thecontribution of Tyr residues in antigen binding); Adib-Conquy et al.(1998) Int. Immunol. 10:341-6 (describing the effect of hydrophobicityin binding) and Beers et al. (2000) Clin. Can. Res. 6:2835-43(describing HCDR3 amino acid mutants). Accordingly, this disclosureprovides an isolated monoclonal antibody, or antigen binding portionthereof, comprising a heavy chain variable region comprising CDR1, CDR2,and CDR3 sequences and a light chain variable region comprising CDR1,CDR2, and CDR3 sequences, wherein:

-   -   (a) the heavy chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequences of SEQ ID NOs: 9-12, and conservative        modifications thereof;    -   (b) the light chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequence of SEQ ID NOs: 21-44, and conservative        modifications thereof; and    -   (c) the antibody binds to native human CXCR4 expressed on a cell        surface.

Additionally or alternatively, the antibody may possess one or more ofthe following functional properties: (i) binds to human CXCR4 with aK_(D) of 1×10⁻⁷ M or less; (ii) inhibits SDF-1 binding to CXCR4; (iii)inhibits SDF-1-induced calcium flux in cells expressing CXCR4; (iv)inhibits SDF-1-induced migration of cells expressing CXCR4; (v) inhibitscapillary tube formation by HuVECs; (vi) induces apoptosis in cellsexpressing CXCR4 in vitro and/or in vivo; (vii) inhibits growth ofCXCR4⁺ tumor cells in vitro and/or in vivo; and/or (viii) inhibitsmetastases of CXCR4⁺ tumor cells.

In a preferred embodiment, the heavy chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 5-8, and conservative modificationsthereof; and the light chain variable region CDR2 sequence comprises anamino acid sequence selected from the group consisting of amino acidsequences of SEQ ID NOs: 17-20, and conservative modifications thereof.In another preferred embodiment, the heavy chain variable region CDR1sequence comprises an amino acid sequence selected from the groupconsisting of amino acid sequences of SEQ ID NOs: 1-4, and conservativemodifications thereof; and the light chain variable region CDR1 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 13-16, and conservativemodifications thereof.

In various embodiments, the antibody can be, for example, humanantibodies, humanized antibodies or chimeric antibodies.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of this disclosure by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody ofthis disclosure can be replaced with other amino acid residues from thesame side chain family and the altered antibody can be tested forretained function (i.e., the functions set forth above) using thefunctional assays described herein.

Antibodies that Bind to the Same Epitope as Anti-CXCR4 Antibodies

In another embodiment, this disclosure provides antibodies that bind tothe same epitope on human CXCR4 as any of the anti-CXCR4 monoclonalantibodies of this disclosure (i.e., antibodies that have the ability tocross-compete for binding to CXCR4 with any of the monoclonal antibodiesof this disclosure). In preferred embodiments, the reference antibodyfor cross-competition studies can be the monoclonal antibody F7 (havingV_(H) and V_(L) sequences as shown in SEQ ID NOs: 25 and 29,respectively), or the monoclonal antibody F9 (having V_(H) and V_(L),sequences as shown in SEQ ID NOs: 26 and 30, respectively) or themonoclonal antibody D1 (having V_(H) and V_(L) sequences as shown in SEQID NOs: 27 and 31, respectively) or the monoclonal antibody E2 (havingV_(H) and V_(L) sequences as shown in SEQ ID NOs: 28 and 32,respectively).

Such cross-competing antibodies can be identified based on their abilityto cross-compete with F7, F9, D1 or E2 in standard CXCR4 binding assays.For example, flow cytometry with CEM cells may be used to demonstratecross-competition with the antibodies of the current disclosure, whereinthe reference antibody is labeled with FITC and the ability of a testantibody to inhibit the binding of the FITC-labeled reference antibodyto CEM cells is evaluated. The ability of a test antibody to inhibit thebinding of, for example, F7, F9, D1 or E2, to human CXCR4 demonstratesthat the test antibody can compete with F7, F9, D1 or E2 for binding tohuman CXCR4 and thus binds to the same epitope on human CXCR4 as F7, F9,D1 or E2. In a preferred embodiment, the antibody that binds to the sameepitope on CXCR4 as F7, F9, D1 or E2 is a human monoclonal antibody.Such human monoclonal antibodies can be prepared and isolated asdescribed in the Examples.

Engineered and Modified Antibodies

An antibody of this disclosure further can be prepared using an antibodyhaving one or more of the V_(H) and/or V_(L) sequences disclosed hereinas starting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e., V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

In certain embodiments, CDR grafting can be used to engineer variableregions of antibodies. Antibodies interact with target antigenspredominantly through amino acid residues that are located in the sixheavy and light chain complementarity determining regions (CDRs). Forthis reason, the amino acid sequences within CDRs are more diversebetween individual antibodies than sequences outside of CDRs. BecauseCDR sequences are responsible for most antibody-antigen interactions, itis possible to express recombinant antibodies that mimic the propertiesof specific naturally occurring antibodies by constructing expressionvectors that include CDR sequences from the specific naturally occurringantibody grafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. etal. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of this disclosure pertains to anisolated monoclonal antibody, or antigen binding portion thereof,comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3sequences comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-4, SEQ ID NOs: 5-8, and SEQ ID NOs: 9-12,respectively, and a light chain variable region comprising CDR1, CDR2,and CDR3 sequences comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 13-16, SEQ ID NOs: 17-20, and SEQ IDNOs: 21-24, respectively. Thus, such antibodies contain the V_(H) andV_(L) CDR sequences of monoclonal antibodies F7, F9, D1 or E2 yet maycontain different framework sequences from these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.(1992) “The Repertoire of Human Germline V_(H) Sequences Reveals aboutFifty Groups of V_(H) Segments with Different Hypervariable Loops” J.Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) “A Directory ofHuman Germ-line V_(H) Segments Reveals a Strong Bias in their Usage”Eur. J. Immunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference. As another example, the germline DNAsequences for human heavy and light chain variable region genes can befound in the Genbank database. For example, the following heavy chaingermline sequences found in the HCo7 HuMAb mouse are available in theaccompanying Genbank accession numbers: 1-69 (NG_(—)0010109,NT_(—)024637 and BC070333), 3-33 (NG_(—)0010109 and NT_(—)024637) and3-7 (NG_(—)0010109 and NT_(—)024637). As another example, the followingheavy chain germline sequences found in the HCo12 HuMAb mouse areavailable in the accompanying Genbank accession numbers: 1-69(NG_(—)0010109, NT_(—)024637 and BC070333), 5-51 (NG_(—)0010109 andNT_(—)024637), 4-34 (NG_(—)0010109 and NT_(—)024637), 3-30.3 (CAJ556644)and 3-23 (AJ406678). Yet another source of human heavy and light chaingermline sequences is the database of human immunoglobulin genesavailable from IMGT (http://imgt.cines.fr).

Antibody protein sequences are compared against a compiled proteinsequence database using one of the sequence similarity searching methodscalled the Gapped BLAST (Altschul et al. (1997) Nucleic Acids Research25:3389-3402), which is well known to those skilled in the art. BLAST isa heuristic algorithm in that a statistically significant alignmentbetween the antibody sequence and the database sequence is likely tocontain high-scoring segment pairs (HSP) of aligned words. Segment pairswhose scores cannot be improved by extension or trimming is called ahit. Briefly, the nucleotide sequences of VBASE origin(http://vhase.mrc-cpe.cam.ac.uk/vbase1/list2.php) are translated and theregion between and including FR1 through FR3 framework region isretained. The database sequences have an average length of 98 residues.Duplicate sequences which are exact matches over the entire length ofthe protein are removed. A BLAST search for proteins using the programblastp with default, standard parameters except the low complexityfilter, which is turned off, and the substitution matrix of BLOSUM62,filters for top 5 hits yielding sequence matches. The nucleotidesequences are translated in all six frames and the frame with no stopcodons in the matching segment of the database sequence is consideredthe potential hit. This is in turn confirmed using the BLAST programtblastx, which translates the antibody sequence in all six frames andcompares those translations to the VBASE nucleotide sequencesdynamically translated in all six frames. Other human germline sequencedatabases, such as that available from IMGT (http://imgt.cines.fr), canbe searched similarly to VBASE as described above.

The identities are exact amino acid matches between the antibodysequence and the protein database over the entire length of thesequence. The positives (identities+substitution match) are notidentical but amino acid substitutions guided by the BLOSUM62substitution matrix. If the antibody sequence matches two of thedatabase sequences with same identity, the hit with most positives wouldbe decided to be the matching sequence hit.

Preferred framework sequences for use in the antibodies of thisdisclosure are those that are structurally similar to the frameworksequences used by selected antibodies of this disclosure, e.g., similarto the V_(H) 3-48 framework sequences (SEQ ID NO: 49) and/or the V_(K)L15 framework sequence (SEQ ID NO: 50) used by preferred monoclonalantibodies of this disclosure. The V_(H) CDR1, CDR2, and CDR3 sequences,and the V_(K) CDR1, CDR2, and CDR3 sequences, can be grafted ontoframework regions that have the identical sequence as that found in thegermline immunoglobulin gene from which the framework sequence derive,or the CDR sequences can be grafted onto framework regions that containone or more mutations as compared to the germline sequences. Forexample, it has been found that in certain instances it is beneficial tomutate residues within the framework regions to maintain or enhance theantigen binding ability of the antibody (see e.g., U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(K) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, can beevaluated in in vitro or in vivo assays as described herein and providedin the Examples. Preferably conservative modifications (as discussedabove) are introduced. The mutations may be amino acid substitutions,additions or deletions, but are preferably substitutions. Moreover,typically no more than one, two, three, four or five residues within aCDR region are altered.

Accordingly, in another embodiment, the instant disclosure providesisolated anti-CXCR4 monoclonal antibodies, or antigen binding portionsthereof, comprising a heavy chain variable region comprising: (a) aV_(H) CDR1 region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1-4, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 1-4; (b) a V_(H) CDR2 regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 5-8, or an amino acid sequence having one, two, three, fouror five amino acid substitutions, deletions or additions as compared toSEQ ID NOs: 5-8; (c) a V_(H) CDR3 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 9-12, or anamino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 9-12;(d) a V_(K) CDR1 region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 13-16, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 13-16; (e) a V_(K) CDR2 regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 17-20, or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 17-20; and (f) a V_(K) CDR3 region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:21-24, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNOs: 21-24.

Engineered antibodies of this disclosure include those in whichmodifications have been made to framework residues within V_(H) and/orV_(K), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived.

For example, for the F7 V_(H) region, the following framework regionamino acid positions (using the Kabat numbering system) differ fromgermline: 1, 6 and 25. One, two or all three of these positions can bebackmutated to germline sequences by making one, two or all three of thefollowing substitutions: Q1E, Q6E and A25S. A preferred modified form ofthe F7 V_(H) region is F7GL (the amino acid sequence of which is shownin FIG. 5A and in SEQ ID NO: 41), which has the following frameworksubstitutions: Q1E and Q6E.

Furthermore, for the F7 V_(k) region, the following framework regionamino acid positions (using the Kabat numbering system) differ fromgermline: 1, 3 and 84. One, two or all three of these positions can bebackmutated to germline sequences by making one, two or all three of thefollowing substitutions: A1D, R3Q and V84A. A preferred modified form ofthe F7 V_(k) region is F7GL V_(k) (the amino acid sequence of which isshown in FIG. 5B and in SEQ ID NO: 45), which has the followingframework substitutions: A1D and R3Q.

Furthermore, for the F9 V_(H) region, the following framework regionamino acid positions (using the Kabat numbering system) differ fromgermline: 1, 6 and 25. One, two or all three of these positions can bebackmutated to germline sequences by making one, two or all three of thefollowing substitutions: Q1E, Q6E and A25S. A preferred modified form ofthe F9 V_(H) region is F9GL V_(H) (the amino acid sequence of which isshown in FIG. 6A and in SEQ ID NO: 42), which has the followingframework substitutions: Q1E and Q6E.

Furthermore, for the F9 V_(k) region, the following framework regionamino acid positions (using the Kabat numbering system) differ fromgermline: 1, 3, 4 and 60. One, two, three or all four of these positionscan be backmutated to germline sequences by making one, two, three orall four of the following substitutions: E1D, V3Q, L4M and P60S. Apreferred modified form of the F9 V_(k) region is F9GL V_(k) (the aminoacid sequence of which is shown in FIG. 6B and in SEQ ID NO: 46), whichhas the following framework substitutions: E1D, V3Q and L4M.

Furthermore, for the D1 V_(H) region, the following framework regionamino acid positions (using the Kabat numbering system) differ fromgermline: 6, 25 and 76. One, two or all three of these positions can bebackmutated to germline sequences by making one, two or all three of thefollowing substitutions: Q6E, A25S and R76K. A preferred modified formof the D1 V_(H) region is D1GL V_(H) (the amino acid sequence of whichis shown in FIG. 7A and in SEQ ID NO: 43), which has the followingframework substitution: Q6E.

Furthermore, for the D1 V_(k) region, the following framework regionamino acid positions (using the Kabat numbering system) differ fromgermline: 1, 3, 4, 45 and 46, One, two, three, four or all five of thesepositions can be backmutated to germline sequences by making one, two,three, four or all five of the following substitutions: V1D, W3Q, V4M,E45K and L46S. A preferred modified form of the D1 V_(k) region is D1GLV_(k) (the amino acid sequence of which is shown in FIG. 7B and in SEQID NO: 47), which has the following framework substitutions: V1D, W3Qand V4M.

Furthermore, for the E2 V_(H) region, the following framework regionamino acid positions (using the Kabat numbering system) differ fromgermline: 6 and 25. One or both of these positions can be backmutated togermline sequences by making one or both of the following substitutions:Q6E and A25S. A preferred modified form of the E2 V_(H) region is E2GLV_(H) (the amino acid sequence of which is shown in FIG. 8A and in SEQID NO: 44), which has the following framework substitution: Q6E.

Furthermore, for the E2 V_(k) region, the following framework regionamino acid positions (using the Kabat numbering system) differ fromgermline: 1, 3 and 4. One, two or all three of these positions can bebackmutated to germline sequences by making one, two or all three of thefollowing substitutions: E1D, V3Q and L4M. A preferred modified form ofthe E2 V_(k) region is E2GL V_(k) (the amino acid sequence of which isshown in FIG. 8B and in SEQ ID NO: 48), which has the followingframework substitutions: E1D, V3Q and L4M.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of this disclosure may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of this disclosure maybe chemically modified (e.g., one or more chemical moieties can beattached to the antibody) or be modified to alter its glycosylation,again to alter one or more functional properties of the antibody. Eachof these embodiments is described in further detail below. The numberingof residues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072 by Presta.Moreover, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII andFcRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334and 339 were shown to improve binding to FcγRIII. Additionally, thefollowing combination mutants were shown to improve FcγRIII binding:T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of this disclosure to thereby produce an antibodywith altered glycosylation. For example, the cell lines Ms704, Ms705,and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6)fucosyltransferase), such that antibodies expressed in the Ms704, Ms705,and Ms709 cell lines lack fucose on their carbohydrates. The Ms704,Ms705, and Ms709 FUT8^(−/−) cell lines were created by the targeteddisruption of the FUT8 gene in CHO/DG44 cells using two replacementvectors (see U.S. Patent Publication No. 20040110704 by Yamane et al.and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As anotherexample, EP 1,176,195 by Hanai et al. describes a cell line with afunctionally disrupted FUT8 gene, which encodes a fucosyl transferase,such that antibodies expressed in such a cell line exhibithypofucosylation by reducing or eliminating the alpha 1,6 bond-relatedenzyme. Hanai et al. also describe cell lines which have a low enzymeactivity for adding fucose to the N-acetylglucosamine that binds to theFc region of the antibody or does not have the enzyme activity, forexample the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT PublicationWO 03/035835 by Presta describes a variant CHO cell line, Lec13 cells,with reduced ability to attach fucose to Asn(297)-linked carbohydrates,also resulting in hypofucosylation of antibodies expressed in that hostcell (see also Shields, R. L. et al. (2002) J. Biol. Chem.277:26733-26740). Antibodies with a modified glycosylation profile canalso be produced in chicken eggs, as described in US Patent ApplicationNo. PCT/US06/05853. Alternatively, antibodies with a modifiedglycosylation profile can be produced in plant cells, such as Lemna.Methods for production of antibodies in a plant system are disclosed inthe U.S. patent application 60/836,998, filed on Aug. 11, 2006. PCTPublication WO 99/54342 by Umana et al. describes cell lines engineeredto express glycoprotein-modifying glycosyl transferases (e.g.,beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).Alternatively, the fucose residues of the antibody may be cleaved offusing a fucosidase enzyme. For example, the fucosidasealpha-L-fucosidase removes fucosyl residues from antibodies (Tarentino,A. L. et al. (1975) Biochem. 14:5516-23).

Another modification of the antibodies herein that is contemplated bythis disclosure is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.Preferably, the pegylation is carried out via an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of this disclosure. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Antibody Fragments and Antibody Mimetics

The instant invention is not limited to traditional antibodies and maybe practiced through the use of antibody fragments and antibodymimetics. As detailed below, a wide variety of antibody fragment andantibody mimetic technologies have now been developed and are widelyknown in the art. While a number of these technologies, such as domainantibodies, Nanobodies, and UniBodies make use of fragments of, or othermodifications to, traditional antibody structures, there are alsoalternative technologies, such as Affibodies, DARPins, Anticalins,Avimers, and Versabodies that employ binding structures that, while theymimic traditional antibody binding, are generated from and function viadistinct mechanisms.

Domain Antibodies (dAbs) are the smallest functional binding units ofantibodies, corresponding to the variable regions of either the heavy(VH) or light (VL) chains of human antibodies. Domain Antibodies have amolecular weight of approximately 13 kDa. Domantis has developed aseries of large and highly functional libraries of fully human VH and VLdAbs (more than ten billion different sequences in each library), anduses these libraries to select dAbs that are specific to therapeutictargets. In contrast to many conventional antibodies, Domain Antibodiesare well expressed in bacterial, yeast, and mammalian cell systems.Further details of domain antibodies and methods of production thereofmay be obtained by reference to U.S. Pat. Nos. 6,291,158; 6,582,915;6,593,081; 6,172,197; 6,696,245; US Serial No. 2004/0110941; Europeanpatent application No. 1433846 and European Patents 0368684 & 0616640;WO05/035572, WO04/101790, WO04/081026, WO04/058821, WO04/003019 andWO03/002609, each of which is herein incorporated by reference in itsentirety.

Nanobodies are antibody-derived therapeutic proteins that contain theunique structural and functional properties of naturally-occurringheavy-chain antibodies. These heavy-chain antibodies contain a singlevariable domain (VHH) and two constant domains (CH2 and CH3).Importantly, the cloned and isolated VHH domain is a perfectly stablepolypeptide harbouring the full antigen-binding capacity of the originalheavy-chain antibody. Nanobodies have a high homology with the VHdomains of human antibodies and can be further humanized without anyloss of activity. Importantly, Nanobodies have a low immunogenicpotential, which has been confirmed in primate studies with Nanobodylead compounds.

Nanobodies combine the advantages of conventional antibodies withimportant features of small molecule drugs. Like conventionalantibodies, Nanobodies show high target specificity, high affinity fortheir target and low inherent toxicity. However, like small moleculedrugs they can inhibit enzymes and readily access receptor clefts.Furthermore, Nanobodies are extremely stable, can be administered bymeans other than injection (see e.g. WO 04/041867, which is hereinincorporated by reference in its entirety) and are easy to manufacture.Other advantages of Nanobodies include recognizing uncommon or hiddenepitopes as a result of their small size, binding into cavities oractive sites of protein targets with high affinity and selectivity dueto their unique 3-dimensional, drug format flexibility, tailoring ofhalf-life and ease and speed of drug discovery.

Nanobodies are encoded by single genes and are efficiently produced inalmost all prokaryotic and eukaryotic hosts e.g. E. coli (see e.g. U.S.Pat. No. 6,765,087, which is herein incorporated by reference in itsentirety), molds (for example Aspergillus or Trichoderma) and yeast (forexample Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see e.g.U.S. Pat. No. 6,838,254, which is herein incorporated by reference inits entirety). The production process is scalable and multi-kilogramquantities of Nanobodies have been produced. Because Nanobodies exhibita superior stability compared with conventional antibodies, they can beformulated as a long shelf-life, ready-to-use solution.

The Nanoclone method (see e.g. WO 06/079372, which is hereinincorporated by reference in its entirety) is a proprietary method forgenerating Nanobodies against a desired target, based on automatedhigh-throughout selection of B-cells and could be used in the context ofthe instant invention.

UniBodies are another antibody fragment technology, however this one isbased upon the removal of the hinge region of IgG4 antibodies. Thedeletion of the hinge region results in a molecule that is essentiallyhalf the size of traditional IgG4 antibodies and has a univalent bindingregion rather than the bivalent binding region of IgG4 antibodies. It isalso well known that IgG4 antibodies are inert and thus do not interactwith the immune system, which may be advantageous for the treatment ofdiseases where an immune response is not desired, and this advantage ispassed onto UniBodies. For example, UniBodies may function to inhibit orsilence, but not kill, the cells to which they are bound. Additionally,UniBody binding to cancer cells do not stimulate them to proliferate.Furthermore, because UniBodies are about half the size of traditionalIgG4 antibodies, they may show better distribution over larger solidtumors with potentially advantageous efficacy. UniBodies are clearedfrom the body at a similar rate to whole IgG4 antibodies and are able tobind with a similar affinity for their antigens as whole antibodies.Further details of UniBodies may be obtained by reference to patentWO2007/059782, which is herein incorporated by reference in itsentirety.

Affibody molecules represent a new class of affinity proteins based on a58-amino acid residue protein domain, derived from one of theIgG-binding domains of staphylococcal protein A. This three helix bundledomain has been used as a scaffold for the construction of combinatorialphagemid libraries, from which Affibody variants that target the desiredmolecules can be selected using phage display technology (Nord K,Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren P A, Bindingproteins selected from combinatorial libraries of an α-helical bacterialreceptor domain, Nat Biotechnol 1997; 15:772-7. Ronmark J, Gronlund H,Uhlen M, Nygren P A, Human immunoglobulin A (IgA)-specific ligands fromcombinatorial engineering of protein A, Eur J Biochem 2002;269:2647-55.). The simple, robust structure of Affibody molecules incombination with their low molecular weight (6 kDa), make them suitablefor a wide variety of applications, for instance, as detection reagents(Ronmark J, Hansson M, Nguyen T, et al, Construction andcharacterization of affibody-Fc chimeras produced in Escherichia coli, JImmunol Methods 2002; 261:199-211) and to inhibit receptor interactions(Sandstorm K, Xu Z, Forsberg G, Nygren P A, Inhibition of the CD28-CD80co-stimulation signal by a CD28-binding Affibody ligand developed bycombinatorial protein engineering, Protein Eng 2003; 16:691-7). Furtherdetails of Affibodies and methods of production thereof may be obtainedby reference to U.S. Pat. No. 5,831,012 which is herein incorporated byreference in its entirety.

Labelled Affibodies may also be useful in imaging applications fordetermining abundance of Isoforms.

DARPins (Designed Ankyrin Repeat Proteins) are one example of anantibody mimetic DRP (Designed Repeat Protein) technology that has beendeveloped to exploit the binding abilities of non-antibody polypeptides.Repeat proteins such as ankyrin or leucine-rich repeat proteins, areubiquitous binding molecules, which occur, unlike antibodies, intra- andextracellularly. Their unique modular architecture features repeatingstructural units (repeats), which stack together to form elongatedrepeat domains displaying variable and modular target-binding surfaces.Based on this modularity, combinatorial libraries of polypeptides withhighly diversified binding specificities can be generated. This strategyincludes the consensus design of self-compatible repeats displayingvariable surface residues and their random assembly into repeat domains.

DARPins can be produced in bacterial expression systems at very highyields and they belong to the most stable proteins known. Highlyspecific, high-affinity DARPins to a broad range of target proteins,including human receptors, cytokines, kinases, human proteases, virusesand membrane proteins, have been selected. DARPins having affinities inthe single-digit nanomolar to picomolar range can be obtained.

DARPins have been used in a wide range of applications, including ELISA,sandwich ELISA, flow cytometric analysis (FACS), immunohistochemistry(IHC), chip applications, affinity purification or Western blotting.DARPins also proved to be highly active in the intracellular compartmentfor example as intracellular marker proteins fused to green fluorescentprotein (GFP). DARPins were further used to inhibit viral entry withIC50 in the pM range. DARPins are not only ideal to blockprotein-protein interactions, but also to inhibit enzymes. Proteases,kinases and transporters have been successfully inhibited, most often anallosteric inhibition mode. Very fast and specific enrichments on thetumor and very favorable tumor to blood ratios make DARPins well suitedfor in vivo diagnostics or therapeutic approaches.

Additional information regarding DARPins and other DRP technologies canbe found in US Patent Application Publication No. 2004/0132028 andInternational Patent Application Publication No. WO 02/20565, both ofwhich are hereby incorporated by reference in their entirety.

Anticalins are an additional antibody mimetic technology, however inthis case the binding specificity is derived from lipocalins, a familyof low molecular weight proteins that are naturally and abundantlyexpressed in human tissues and body fluids. Lipocalins have evolved toperform a range of functions in vivo associated with the physiologicaltransport and storage of chemically sensitive or insoluble compounds.Lipocalins have a robust intrinsic structure comprising a highlyconserved β-barrel which supports four loops at one terminus of theprotein. These loops form the entrance to a binding pocket andconformational differences in this part of the molecule account for thevariation in binding specificity between individual lipocalins.

While the overall structure of hypervariable loops supported by aconserved β-sheet framework is reminiscent of immunoglobulins,lipocalins differ considerably from antibodies in terms of size, beingcomposed of a single polypeptide chain of 160-180 amino acids which ismarginally larger than a single immunoglobulin domain.

Lipocalins are cloned and their loops are subjected to engineering inorder to create Anticalins. Libraries of structurally diverse Anticalinshave been generated and Anticalin display allows the selection andscreening of binding function, followed by the expression and productionof soluble protein for further analysis in prokaryotic or eukaryoticsystems. Studies have successfully demonstrated that Anticalins can bedeveloped that are specific for virtually any human target protein canbe isolated and binding affinities in the nanomolar or higher range canbe obtained.

Anticalins can also be formatted as dual targeting proteins, so-calledDuocalins. A Duocalin binds two separate therapeutic targets in oneeasily produced monomeric protein using standard manufacturing processeswhile retaining target specificity and affinity regardless of thestructural orientation of its two binding domains.

Modulation of multiple targets through a single molecule is particularlyadvantageous in diseases known to involve more than a single causativefactor. Moreover, bi- or multivalent binding formats such as Duocalinshave significant potential in targeting cell surface molecules indisease, mediating agonistic effects on signal transduction pathways orinducing enhanced internalization effects via binding and clustering ofcell surface receptors. Furthermore, the high intrinsic stability ofDuocalins is comparable to monomeric Anticalins, offering flexibleformulation and delivery potential for Duocalins.

Additional information regarding Anticalins can be found in U.S. Pat.No. 7,250,297 and International Patent Application Publication No. WO99/16873, both of which are hereby incorporated by reference in theirentirety.

Another antibody mimetic technology useful in the context of the instantinvention are Avimers. Avimers are evolved from a large family of humanextracellular receptor domains by in vitro exon shuffling and phagedisplay, generating multidomain proteins with binding and inhibitoryproperties. Linking multiple independent binding domains has been shownto create avidity and results in improved affinity and specificitycompared with conventional single-epitope binding proteins. Otherpotential advantages include simple and efficient production ofmultitarget-specific molecules in Escherichia coli, improvedthermostability and resistance to proteases. Avimers with sub-nanomolaraffinities have been obtained against a variety of targets.

Additional information regarding Avimers can be found in US PatentApplication Publication Nos. 2006/0286603, 2006/0234299, 2006/0223114,2006/0177831, 2006/0008844, 2005/0221384, 2005/0164301, 2005/0089932,2005/0053973, 2005/0048512, 2004/0175756, all of which are herebyincorporated by reference in their entirety.

Versabodies are another antibody mimetic technology that could be usedin the context of the instant invention. Versabodies are small proteinsof 3-5 kDa with >15% cysteines, which form a high disulfide densityscaffold, replacing the hydrophobic core that typical proteins have. Thereplacement of a large number of hydrophobic amino acids, comprising thehydrophobic core, with a small number of disulfides results in a proteinthat is smaller, more hydrophilic (less aggregation and non-specificbinding), more resistant to proteases and heat, and has a lower densityof T-cell epitopes, because the residues that contribute most to MHCpresentation are hydrophobic. All four of these properties arewell-known to affect immunogenicity, and together they are expected tocause a large decrease in immunogenicity.

The inspiration for Versabodies comes from the natural injectablebiopharmaceuticals produced by leeches, snakes, spiders, scorpions,snails, and anemones, which are known to exhibit unexpectedly lowimmunogenicity. Starting with selected natural protein families, bydesign and by screening the size, hydrophobicity, proteolytic antigenprocessing, and epitope density are minimized to levels far below theaverage for natural injectable proteins.

Given the structure of Versabodies, these antibody mimetics offer aversatile format that includes multi-valency, multi-specificity, adiversity of half-life mechanisms, tissue targeting modules and theabsence of the antibody Fc region. Furthermore, Versabodies aremanufactured in E. coli at high yields, and because of theirhydrophilicity and small size, Versabodies are highly soluble and can beformulated to high concentrations. Versabodies are exceptionally heatstable (they can be boiled) and offer extended shelf-life.

Additional information regarding Versabodies can be found in US PatentApplication Publication No. 2007/0191272 which is hereby incorporated byreference in its entirety.

The detailed description of antibody fragment and antibody mimetictechnologies provided above is not intended to be a comprehensive listof all technologies that could be used in the context of the instantspecification. For example, and also not by way of limitation, a varietyof additional technologies including alternative polypeptide-basedtechnologies, such as fusions of complimentary determining regions asoutlined in Qui et al., Nature Biotechnology, 25(8) 921-929 (2007),which is hereby incorporated by reference in its entirety, as well asnucleic acid-based technologies, such as the RNA aptamer technologiesdescribed in U.S. Pat. Nos. 5,789,157, 5,864,026, 5,712,375, 5,763,566,6,013,443, 6,376,474, 6,613,526, 6,114,120, 6,261,774, and 6,387,620,all of which are hereby incorporated by reference, could be used in thecontext of the instant invention.

Antibody Physical Properties

The antibodies of the present disclosure may be further characterized bythe various physical properties of the anti-CXCR4 antibodies. Variousassays may be used to detect and/or differentiate different classes ofantibodies based on these physical properties.

In some embodiments, antibodies of the present disclosure may containone or more glycosylation sites in either the light or heavy chainvariable region. The presence of one or more glycosylation sites in thevariable region may result in increased immunogenicity of the antibodyor an alteration of the pK of the antibody due to altered antigenbinding (Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala F A andMorrison S L (2004) J Immunol 172:5489-94; Wallick et al (1988) J ExpMed 168:1099-109; Spiro R G (2002) Glycobiology 12:43R-56R; Parekh et al(1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).Glycosylation has been known to occur at motifs containing an N-X-S/Tsequence. Variable region glycosylation may be tested using a Glycoblotassay, which cleaves the antibody to produce a Fab, and then tests forglycosylation using an assay that measures periodate oxidation andSchiff base formation. Alternatively, variable region glycosylation maybe tested using Dionex light chromatography (Dionex-LC), which cleavessaccharides from a Fab into monosaccharides and analyzes the individualsaccharide content. In some instances, it is preferred to have ananti-CXCR4 antibody that does not contain variable region glycosylation.This can be achieved either by selecting antibodies that do not containthe glycosylation motif in the variable region or by mutating residueswithin the glycosylation motif using standard techniques well known inthe art.

In a preferred embodiment, the antibodies of the present disclosure donot contain asparagine isomerism sites. A deamidation or isoasparticacid effect may occur on N-G or D-G sequences, respectively. Thedeamidation or isoaspartic acid effect results in the creation ofisoaspartic acid which decreases the stability of an antibody bycreating a kinked structure off a side chain carboxy terminus ratherthan the main chain. The creation of isoaspartic acid can be measuredusing an iso-quant assay, which uses a reverse-phase HPLC to test forisoaspartic acid.

Each antibody will have a unique isoelectric point (pI), but generallyantibodies will fall in the pH range of between 6 and 9.5. The pI for anIgG1 antibody typically falls within the pH range of 7-9.5 and the pIfor an IgG4 antibody typically falls within the pH range of 6-8.Antibodies may have a pI that is outside this range. Although theeffects are generally unknown, there is speculation that antibodies witha pI outside the normal range may have some unfolding and instabilityunder in vivo conditions. The isoelectric point may be tested using acapillary isoelectric focusing assay, which creates a pH gradient andmay utilize laser focusing for increased accuracy (Janini et al (2002)Electrophoresis 23:1605-11; Ma et al. (2001) Chromatographia 53:S75-89;Hunt et al (1998) J. Chromatogr A 800:355-67). In some instances, it ispreferred to have an anti-CXCR4 antibody that contains a pI value thatfalls in the normal range. This can be achieved either by selectingantibodies with a pI in the normal range, or by mutating charged surfaceresidues using standard techniques well known in the art.

Each antibody will have a melting temperature that is indicative ofthermal stability (Krishnamurthy R and Manning M C (2002) Curr PharmBiotechnol 3:361-71). A higher thermal stability indicates greateroverall antibody stability in vivo. The melting point of an antibody maybe measure using techniques such as differential scanning calorimetry(Chen et al. (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) ImmunolLett 68:47-52). T_(M1) indicates the temperature of the initialunfolding of the antibody. T_(M2) indicates the temperature of completeunfolding of the antibody. Generally, it is preferred that the T_(M1) ofan antibody of the present disclosure is greater than 60° C., preferablygreater than 65° C., even more preferably greater than 70° C.Alternatively, the thermal stability of an antibody may be measure usingcircular dichroism (Murray et al (2002) J. Chromatogr Sci 40:343-9).

In a preferred embodiment, antibodies are selected that do not rapidlydegrade. Fragmentation of an anti-CXCR4 antibody may be measured usingcapillary electrophoresis (CE) and MALDI-MS, as is well understood inthe art (Alexander A J and Hughes D E (1995) Anal Chem 67:3626-32).

In another preferred embodiment, antibodies are selected that haveminimal aggregation effects. Aggregation may lead to triggering of anunwanted immune response and/or altered or unfavorable pharmacokineticproperties. Generally, antibodies are acceptable with aggregation of 25%or less, preferably 20% or less, even more preferably 15% or less, evenmore preferably 10% or less and even more preferably 5% or less.Aggregation may be measured by several techniques well known in the art,including size-exclusion column (SEC) high performance liquidchromatography (HPLC), and light scattering to identify monomers,dimers, trimers or multimers.

Methods of Engineering Antibodies

As discussed above, the anti-CXCR4 antibodies having V_(H) and V_(K)sequences disclosed herein can be used to create new anti-CXCR4antibodies by modifying the V_(H) and/or V_(K) sequences, or theconstant region(s) attached thereto. Thus, in another aspect of thisdisclosure, the structural features of an anti-CXCR4 antibody of thisdisclosure, e.g. F7, F9, D1 or E2, are used to create structurallyrelated anti-CXCR4 antibodies that retain at least one functionalproperty of the antibodies of this disclosure, such as binding to humanCXCR4. For example, one or more CDR regions of F7, F9, D1 or E2, ormutations thereof, can be combined recombinantly with known frameworkregions and/or other CDRs to create additional,recombinantly-engineered, anti-CXCR4 antibodies of this disclosure, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the V_(H) and/or V_(K) sequences provided herein, orone or more CDR regions thereof. To create the engineered antibody, itis not necessary to actually prepare (i.e., express as a protein) anantibody having one or more of the V_(H) and/or V_(K) sequences providedherein, or one or more CDR regions thereof. Rather, the informationcontained in the sequence(s) is used as the starting material to createa “second generation” sequence(s) derived from the original sequence(s)and then the “second generation” sequence(s) is prepared and expressedas a protein.

Accordingly, in another embodiment, this disclosure provides a methodfor preparing an anti-CXCR4 antibody comprising:

-   -   (a) providing: (i) a heavy chain variable region antibody        sequence comprising a CDR1 sequence selected from the group        consisting of SEQ ID NOs: 1-4, a CDR2 sequence selected from the        group consisting of SEQ ID NOs: 5-8, and/or a CDR3 sequence        selected from the group consisting of SEQ ID NOs: 9-12;        and/or (ii) a light chain variable region antibody sequence        comprising a CDR1 sequence selected from the group consisting of        SEQ ID NOs: 13-16, a CDR2 sequence selected from the group        consisting of SEQ ID NOs: 17-20, and/or a CDR3 sequence selected        from the group consisting of SEQ ID NOs: 21-24;    -   (b) altering at least one amino acid residue within the heavy        chain variable region antibody sequence and/or the light chain        variable region antibody sequence to create at least one altered        antibody sequence; and    -   (c) expressing the altered antibody sequence as a protein.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence.

Preferably, the antibody encoded by the altered antibody sequence(s) isone that retains one, some or all of the functional properties of theanti-CXCR4 antibodies described herein, which functional propertiesinclude, but are not limited to:

-   -   (i) binding to native human CXCR4 expressed on a cell surface;    -   (ii) inhibiting binding of SDF-1 to CXCR4;    -   (iii) inhibiting SDF-1-induced calcium flux in cells expressing        CXCR4;    -   (iv) inhibiting SDF-1-induced migration of cells expressing        CXCR4    -   (v) inhibiting capillary tube formation by HuVECs;    -   (vi) binding to human CXCR4 with a K_(D) of 1×10⁻⁷ M or less;    -   (vii) inducing apoptosis in cells expressing CXCR4;    -   (viii) inhibiting tumor cell proliferation in vitro;    -   (ix) inhibiting tumor cell proliferation and/or inducing tumor        cell apoptosis in vivo;    -   (x) inhibiting metastases of CXCR4⁺ tumor cells; and/or    -   (xi) increasing survival time of a CXCR4⁺ tumor-bearing subject.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., flow cytometry, bindingassays, functional assays).

In certain embodiments of the methods of engineering antibodies of thisdisclosure, mutations can be introduced randomly or selectively alongall or part of an anti-CXCR4 antibody coding sequence and the resultingmodified anti-CXCR4 antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art. For example, PCT Publication WO02/092780 by Short describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

Nucleic Acid Molecules Encoding Antibodies of this Disclosure

Another aspect of this disclosure pertains to nucleic acid moleculesthat encode the antibodies of this disclosure. The nucleic acids may bepresent in whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. (1987) Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York. Anucleic acid of this disclosure can be, for example, DNA or RNA and mayor may not contain intronic sequences. In a preferred embodiment, thenucleic acid is a cDNA molecule.

Nucleic acids of this disclosure can be obtained using standardmolecular biology techniques. For antibodies expressed by hybridomas(e.g., hybridomas prepared from transgenic mice carrying humanimmunoglobulin genes as described further below), cDNAs encoding thelight and heavy chains of the antibody made by the hybridoma can beobtained by standard PCR amplification or cDNA cloning techniques. Forantibodies obtained from an immunoglobulin gene library (e.g., usingphage display techniques), a nucleic acid encoding such antibodies canbe recovered from the gene library.

Preferred nucleic acids molecules of this disclosure are those encodingthe V_(H) and V_(L) sequences of the F7, F9, D1 and E2 monoclonalantibodies. DNA sequences encoding the V_(H) sequences of F7, F9, D1 andE2 are shown in SEQ ID NOs: 33-36, respectively. DNA sequences encodingthe V_(L) sequences of F7, F9, D1 and E2 are shown in SEQ ID NOs: 37-40,respectively.

Once DNA fragments encoding V_(H) and V_(L) segments are obtained, theseDNA fragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the V_(H)-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. In preferred embodiments,the light chain constant region can be a kappa or lambda constantregion.

To create a scFv gene, the V_(H)- and V_(L) encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the V_(L) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature348:552-554).

Production of Monoclonal Antibodies of this Disclosure

Monoclonal antibodies (mAbs) of the present disclosure can be producedby a variety of techniques, including conventional monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein (1975) Nature 256: 495. Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibody can be employed e.g., viral oroncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present disclosure can beprepared based on the sequence of a non-human monoclonal antibodyprepared as described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the non-human hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,murine CDR regions can be inserted into a human framework using methodsknown in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S.Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen etal.).

In a preferred embodiment, the antibodies of this disclosure are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstCXCR4 can be generated using transgenic or transchromosomic micecarrying parts of the human immune system rather than the mouse system.These transgenic and transchromosomic mice include mice referred toherein as the HuMAb Mouse® and KM Mouse®, respectively, and arecollectively referred to herein as “human Ig mice.”

The HuMAb Mouse® (Medarex®, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed inLonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101;Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, andHarding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546).Preparation and use of the HuMAb Mouse®, and the genomic modificationscarried by such mice, is further described in Taylor, L. et al. (1992)Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993)International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl.Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al.(1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) InternationalImmunology 6: 579-591; and Fishwild, D. et al. (1996) NatureBiotechnology 14: 845-851, the contents of all of which are herebyspecifically incorporated by reference in their entirety. See further,U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650;5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all toLonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCTPublication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT PublicationNo. WO 01/14424 to Korman et al.

In another embodiment, human antibodies of this disclosure can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. This mouse isreferred to herein as a “KM Mouse®,” and is described in detail in PCTPublication WO 02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-CXCR4 antibodies of this disclosure. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused; such mice are described in, for example, U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-CXCR4 antibodies of this disclosure. For example, mice carryingboth a human heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (e.g., Kuroiwa et al.(2002) Nature Biotechnology 20:889-894 and PCT application No. WO2002/092812) and can be used to raise anti-CXCR4 antibodies of thisdisclosure.

Human monoclonal antibodies of this disclosure can also be preparedusing phage display methods for screening libraries of humanimmunoglobulin genes. Such phage display methods for isolating humanantibodies are established in the art. See for example: U.S. Pat. Nos.5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos.5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404;6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies of this disclosure can also be preparedusing SCID mice into which human immune cells have been reconstitutedsuch that a human antibody response can be generated upon immunization.Such mice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

In a particularly preferred embodiment, human anti-CXCR4 antibodies areprepared using a combination of human Ig mouse and phage displaytechniques, as described in U.S. Pat. No. 6,794,132 by Buechler et al.More specifically, the method first involves raising an anti-CXCR4antibody response in a human Ig mouse (such as a HuMab mouse or KM mouseas described above) by immunizing the mouse with a CXCR4 antigen,followed by isolating nucleic acids encoding human antibody chains fromlymphatic cells of the mouse and introducing these nucleic acids into adisplay vector (e.g., phage) to provide a library of display packages.Thus, each library member comprises a nucleic acid encoding a humanantibody chain and each antibody chain is displayed from the displaypackage. The library then is screened with a CXCR4 antigen to isolatelibrary members that specifically bind CXCR4. Nucleic acid inserts ofthe selected library members then are isolated and sequenced by standardmethods to determine the light and heavy chain variable sequences of theselected CXCR4 binders. The variable regions can be converted tofull-length antibody chains by standard recombinant DNA techniques, suchas cloning of the variable regions into an expression vector thatcarries the human heavy and light chain constant regions such that theVH region is operatively linked to the CH region and the VL region isoperatively linked to the CL region. For a further description of thepreparation of human anti-CXCR4 antibodies using this combinedtransgenic mouse/phage display system, see Example 1.

Immunization of Human Ig Mice

When human Ig mice are used to raise human antibodies of thisdisclosure, such mice can be immunized with a purified or enrichedpreparation of CXCR4 antigen and/or recombinant CXCR4, or cellsexpressing CXCR4, or a CXCR4 fusion protein, as described by Lonberg, N.et al. (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996)Nature Biotechnology 14: 845-851; and PCT Publication WO 98/24884 and WO01/14424. Preferably, the mice will be 6-16 weeks of age upon the firstinfusion. For example, a purified or recombinant preparation (5-50 μg)of CXCR4 antigen can be used to immunize the human Ig miceintraperitoneally. Most preferably, the immunogen used to raise theantibodies of this disclosure comprises human CXCR4 in its nativeconformation within a membrane, non-limiting examples of which includecells transfected to express CXCR4 on their cell surface, cells thatnatively express CXCR4 (e.g., CEM cells), and artificial membranes(e.g., liposomes) into which CXCR4 has been incorporated, such asmagnetic proteoliposomes (MPLs) that incorporate CXCR4 (describedfurther in Example 1).

Detailed procedures to generate fully human monoclonal antibodies toCXCR4 are described in Example 1 below. Cumulative experience withvarious antigens has shown that the transgenic mice respond wheninitially immunized intraperitoneally (IP) with antigen in completeFreund's adjuvant, followed by every other week IP immunizations (up toa total of 6) with antigen in incomplete Freund's adjuvant. However,adjuvants other than Freund's are also found to be effective. Inaddition, whole cells in the absence of adjuvant are found to be highlyimmunogenic. The immune response can be monitored over the course of theimmunization protocol with plasma samples being obtained by retroorbitalbleeds. The plasma can be screened by ELISA (as described below), andmice with sufficient titers of anti-CXCR4 human immunoglobulin can beused for fusions. Mice can be boosted intravenously with antigen 3 daysbefore sacrifice and removal of the spleen. It is expected that 2-3fusions for each immunization may need to be performed. Between 6 and 24mice are typically immunized for each antigen. Usually both HCo7 andHCo12 strains are used. In addition, both HCo7 and HCo12 transgene canbe bred together into a single mouse having two different human heavychain transgenes (HCo7/HCo12). Alternatively or additionally, the KMMouse® strain can be used, as described in Example 1.

Generation of Hybridomas Producing Human Monoclonal Antibodies of thisDisclosure

To generate hybridomas producing human monoclonal antibodies of thisdisclosure, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toone-sixth the number of P3×63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Alternatively, the single cell suspensionof splenic lymphocytes from immunized mice can be fused using anelectric field based electrofusion method, using a CytoPulse largechamber cell fusion electroporator (CytoPulse Sciences, Inc., GlenBurnie Md.). Cells are plated at approximately 2×10⁵ in flat bottommicrotiter plate, followed by a two week incubation in selective mediumcontaining 20% fetal Clone Serum, 18% “653” conditioned media, 5% origen(IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50mg/ml gentamycin and 1×HAT (Sigma; the HAT is added 24 hours after thefusion). After approximately two weeks, cells can be cultured in mediumin which the HAT is replaced with HT. Individual wells can then bescreened by ELISA for human monoclonal IgM and IgG antibodies. Onceextensive hybridoma growth occurs, medium can be observed usually after10-14 days. The antibody secreting hybridomas can be replated, screenedagain, and if still positive for human IgG, the monoclonal antibodiescan be subcloned at least twice by limiting dilution. The stablesubclones can then be cultured in vitro to generate small amounts ofantibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies of thisDisclosure

Antibodies of this disclosure also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the C_(H) segment(s) within the vectorand the V_(K) segment is operatively linked to the C_(L) segment withinthe vector. Additionally or alternatively, the recombinant expressionvector can encode a signal peptide that facilitates secretion of theantibody chain from a host cell. The antibody chain gene can be clonedinto the vector such that the signal peptide is linked in-frame to theamino terminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of this disclosure carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or β-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRα promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of this disclosure may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection). For expression of the light and heavy chains, the expressionvector(s) encoding the heavy and light chains is transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. Although it istheoretically possible to express the antibodies of this disclosure ineither prokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof this disclosure include Chinese Hamster Ovary (CHO cells) (includingdhfr⁻ CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl.Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g.,as described in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462 (to Wilson), WO89/01036 (to Bebbington) and EP 338,841 (to Bebbington). Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Characterization of Antibody Binding to Antigen

Antibodies of this disclosure can be tested for binding to CXCR4 by, forexample, standard flow cytometry methods. Since the antibodies of thisdisclosure preferably recognize CXCR4 in its native conformation withina membrane, testing for binding to CXCR4 preferably is done with anassay (e.g., flow cytometry) that utilizes a reagent expressing nativeconformation CXCR4. Nonlimiting examples of reagents expressing nativeconformation CXCR4 that can be used in the binding assays include cellsthat naturally express CXCR4 (e.g., CEM cells), cells that have beentransfected to express CXCR4 (e.g., R1610 cells transfected with a CXCR4expression vector) and liposomes into which CXCR4 has been incorporated(e.g., magnetic proteoliposomes incorporating CXCR4), each of which isdescribed in further detail in the Examples. Briefly, for the flowcytometry assay, cells expressing CXCR4 are incubated with the testantibody, washed, incubated with a labeled secondary reagent capable ofbinding to the test antibody, washed again, and subjected to analysis todetect the binding of the secondary reagent to the cells (e.g., using aFACS machine). Preferably, mice that develop the highest titers asevaluated by flow cytometry will be used for fusions or for furtherselection of antibodies (e.g., by phage display screening of antibodylibraries made from B cells of the mouse).

A flow cytometry assay as described above can also be used to screen forhybridomas that show positive reactivity with CXCR4 immunogen.Hybridomas expressing antibodies that bind with high avidity to CXCR4are subcloned and further characterized. One clone from each hybridoma,which retains the reactivity of the parent cells (by flow cytometry),can be chosen for making a 5-10 vial cell bank stored at −140° C., andfor antibody purification.

To purify anti-CXCR4 antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-CXCR4 monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Competition studies usingunlabeled monoclonal antibodies and biotinylated monoclonal antibodiescan be performed using a whole cell ELISA assay in which ELISA platesare coated with cells expressing CXCR4, and the ability of the unlabeledantibody to compete with the biotinylated antibody for binding to theCXCR4-expressing cells is examined. Biotinylated mAb binding can bedetected with a strep-avidin-alkaline phosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype. For example, to determine the isotype of a human monoclonalantibody, wells of microtiter plates can be coated with 1 μg/ml ofanti-human immunoglobulin overnight at 4° C. After blocking with 1% BSA,the plates are reacted with 1 μg/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-conjugated probes. Plates aredeveloped and analyzed as described above.

Anti-CXCR4 human IgGs can be further tested for reactivity with CXCR4antigen by Western blotting. Briefly, CXCR4 can be prepared andsubjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis.After electrophoresis, the separated antigens are transferred tonitrocellulose membranes, blocked with 10% fetal calf serum, and probedwith the monoclonal antibodies to be tested. Human IgG binding can bedetected using anti-human IgG alkaline phosphatase and developed withBCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

The binding specificity of an antibody of this disclosure may also bedetermined by monitoring binding of the antibody to cells expressingCXCR4, for example by flow cytometry. Typically, a cell line, such as aCHO cell line, may be transfected with an expression vector encoding atransmembrane form of CXCR4. The transfected protein may comprise a tag,such as a myc-tag, preferably at the N-terminus, for detection using anantibody to the tag. Binding of an antibody of this disclosure to CXCR4may be determined by incubating the transfected cells with the antibody,and detecting bound antibody. Binding of an antibody to the tag on thetransfected protein may be used as a positive control.

Immunoconjugates

In another aspect, the present disclosure features an anti-CXCR4antibody, or a fragment thereof, conjugated to a therapeutic moiety,such as a cytotoxin, a drug (e.g., an immunosuppressant) or aradiotoxin. Such conjugates are referred to herein as“immunoconjugates”. Immunoconjugates that include one or more cytotoxinsare referred to as “immunotoxins.” A cytotoxin or cytotoxic agentincludes any agent that is detrimental to (e.g., kills) cells. Examplesinclude taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents also include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can beconjugated to an antibody of this disclosure include duocarmycins,calicheamicins, maytansines and auristatins, and derivatives thereof. Anexample of a calicheamicin antibody conjugate is commercially available(Mylotarg®; American Home Products).

Cytotoxins can be conjugated to antibodies of this disclosure usinglinker technology available in the art. Examples of linker types thathave been used to conjugate a cytotoxin to an antibody include, but arenot limited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.(2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al. (2003)Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I.and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089-1091;Senter, P. D. and Springer, C. J. (2001) Adv. Drug Deliv. Rev.53:247-264.

Antibodies of the present disclosure also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰ and lutetium¹⁷⁷. Method for preparing radioimmunconjugates areestablished in the art. Examples of radioimmunoconjugates arecommercially available, including Zevalin® (IDEC Pharmaceuticals) andBexxar® (Corixa Pharmaceuticals), and similar methods can be used toprepare radioimmunoconjugates using the antibodies of this disclosure.

The antibody conjugates of this disclosure can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., Immunol.Rev., 62:119-58 (1982).

Bispecific Molecules

In another aspect, the present disclosure features bispecific moleculescomprising an anti-CXCR4 antibody, or a fragment thereof, of thisdisclosure. An antibody of this disclosure, or antigen-binding portionsthereof, can be derivatized or linked to another functional molecule,e.g., another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. The antibody of thisdisclosure may in fact be derivatized or linked to more than one otherfunctional molecule to generate multispecific molecules that bind tomore than two different binding sites and/or target molecules; suchmultispecific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific molecule ofthis disclosure, an antibody of this disclosure can be functionallylinked (e.g., by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other binding molecules, suchas another antibody, antibody fragment, peptide or binding mimetic, suchthat a bispecific molecule results.

Accordingly, the present disclosure includes bispecific moleculescomprising at least one first binding specificity for CXCR4 and a secondbinding specificity for a second target epitope. In a particularembodiment of this disclosure, the second target epitope is an Fcreceptor, e.g., human FcγRI (CD64) or a human Fcα receptor (CD89).Therefore, this disclosure includes bispecific molecules capable ofbinding both to FcγR or FcαR expressing effector cells (e.g., monocytes,macrophages or polymorphonuclear cells (PMNs)), and to target cellsexpressing CXCR4. These bispecific molecules target CXCR4 expressingcells to effector cell and trigger Fc receptor-mediated effector cellactivities, such as phagocytosis of CXCR4 expressing cells, antibodydependent cell-mediated cytotoxicity (ADCC), cytokine release, orgeneration of superoxide anion.

In an embodiment of this disclosure in which the bispecific molecule ismultispecific, the molecule can further include a third bindingspecificity, in addition to an anti-Fc binding specificity and ananti-CXCR4 binding specificity. In one embodiment, the third bindingspecificity is an anti-enhancement factor (EF) portion, e.g., a moleculewhich binds to a surface protein involved in cytotoxic activity andthereby increases the immune response against the target cell. The“anti-enhancement factor portion” can be an antibody, functionalantibody fragment or a ligand that binds to a given molecule, e.g., anantigen or a receptor, and thereby results in an enhancement of theeffect of the binding determinants for the Fc receptor or target cellantigen. The “anti-enhancement factor portion” can bind an Fc receptoror a target cell antigen. Alternatively, the anti-enhancement factorportion can bind to an entity that is different from the entity to whichthe first and second binding specificities bind. For example, theanti-enhancement factor portion can bind a cytotoxic T-cell (e.g. viaCD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that resultsin an increased immune response against the target cell).

In one embodiment, the bispecific molecules of this disclosure compriseas a binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, Fd, dAb or a singlechain Fv. The antibody may also be a light chain or heavy chain dimer,or any minimal fragment thereof such as a Fv or a single chain constructas described in U.S. Pat. No. 4,946,778 to Ladner et al., the contentsof which is expressly incorporated by reference.

In one embodiment, the binding specificity for an Fcγ receptor isprovided by a monoclonal antibody, the binding of which is not blockedby human immunoglobulin G (IgG). As used herein, the term “IgG receptor”refers to any of the eight γ-chain genes located on chromosome 1. Thesegenes encode a total of twelve transmembrane or soluble receptorisoforms which are grouped into three Fcγ receptor classes: FcγRI(CD64), FcγRII(CD32), and FcγRIII (CD16). In one preferred embodiment,the Fcγ receptor a human high affinity FcγRI. The human FcγRI is a 72kDa molecule, which shows high affinity for monomeric IgG (10⁸-10⁹ M⁻¹).

The production and characterization of certain preferred anti-Fcγmonoclonal antibodies are described in PCT Publication WO 88/00052 andin U.S. Pat. No. 4,954,617 to Fanger et al., the teachings of which arefully incorporated by reference herein. These antibodies bind to anepitope of FcγRI, FcγRII or FcγRIII at a site which is distinct from theFcγ binding site of the receptor and, thus, their binding is not blockedsubstantially by physiological levels of IgG. Specific anti-FcγRIantibodies useful in this disclosure are mAb 22, mAb 32, mAb 44, mAb 62and mAb 197. The hybridoma producing mAb 32 is available from theAmerican Type Culture Collection, ATCC Accession No. HB9469. In otherembodiments, the anti-Fcγ receptor antibody is a humanized form ofmonoclonal antibody 22 (H22). The production and characterization of theH22 antibody is described in Graziano, R. F. et al. (1995) J. Immunol155 (10): 4996-5002 and PCT Publication WO 94/10332 to Tempest et al.The H22 antibody producing cell line was deposited at the American TypeCulture Collection under the designation HA022CL1 and has the accessionno. CRL 11177.

In still other preferred embodiments, the binding specificity for an Fcreceptor is provided by an antibody that binds to a human IgA receptor,e.g., an Fc-alpha receptor (FcαRI (CD89)), the binding of which ispreferably not blocked by human immunoglobulin A (IgA). The term “IgAreceptor” is intended to include the gene product of one α-gene (FcαRI)located on chromosome 19. This gene is known to encode severalalternatively spliced transmembrane isoforms of 55 to 110 kDa. FcαRI(CD89) is constitutively expressed on monocytes/macrophages,eosinophilic and neutrophilic granulocytes, but not on non-effector cellpopulations. FcαRI has medium affinity (≈5×10⁷ M⁻¹) for both IgA1 andIgA2, which is increased upon exposure to cytokines such as G-CSF orGM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology16:423-440). Four FcαRI-specific monoclonal antibodies, identified asA3, A59, A62 and A77, which bind FcαRI outside the IgA ligand bindingdomain, have been described (Monteiro, R. C. et al. (1992) J. Immunol.148:1764).

FcαRI and FcγRI are preferred trigger receptors for use in thebispecific molecules of this disclosure because they are (1) expressedprimarily on immune effector cells, e.g., monocytes, PMNs, macrophagesand dendritic cells; (2) expressed at high levels (e.g., 5,000-100,000per cell); (3) mediators of cytotoxic activities (e.g., ADCC,phagocytosis); and (4) mediate enhanced antigen presentation ofantigens, including self-antigens, targeted to them.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules of this disclosure aremurine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present disclosure can be prepared byconjugating the constituent binding specificities, e.g., the anti-FcRand anti-CXCR4 binding specificities, using methods known in the art.For example, each binding specificity of the bispecific molecule can begenerated separately and then conjugated to one another. When thebinding specificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83, and Glennie et al.(1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand×Fab fusion protein. A bispecific molecule of thisdisclosure can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. Nos.5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;5,013,653; 5,258,498; and 5,482,858, all of which are expresslyincorporated herein by reference.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a γ counter or a scintillationcounter or by autoradiography.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a composition, e.g.,a pharmaceutical composition, containing one or a combination ofmonoclonal antibodies, or antigen-binding portion(s) thereof, of thepresent disclosure, formulated together with a pharmaceuticallyacceptable carrier. Such compositions may include one or a combinationof (e.g., two or more different) antibodies, or immunoconjugates orbispecific molecules of this disclosure. For example, a pharmaceuticalcomposition of this disclosure can comprise a combination of antibodies(or immunoconjugates or bispecifics) that bind to different epitopes onthe target antigen or that have complementary activities.

Pharmaceutical compositions of this disclosure also can be administeredin combination therapy, i.e., combined with other agents. For example,the combination therapy can include an anti-CXCR4 antibody of thepresent disclosure combined with at least one other anti-inflammatory orimmunosuppressant agent. Examples of therapeutic agents that can be usedin combination therapy are described in greater detail below in thesection on uses of the antibodies of this disclosure.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjugate, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds of this disclosure may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of this disclosure also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of this disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthis disclosure is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of this disclosure are dictated by anddirectly dependent on (a) the unique characteristics of the activecompound and the particular therapeutic effect to be achieved, and (b)the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Preferred dosage regimens for an anti-CXCR4antibody of this disclosure include 1 mg/kg body weight or 3 mg/kg bodyweight via intravenous administration, with the antibody being givenusing one of the following dosing schedules: (i) every four weeks forsix dosages, then every three months; (ii) every three weeks; (iii) 3mg/kg body weight once followed by 1 mg/kg body weight every threeweeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml.

Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present disclosure may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentdisclosure employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-CXCR4 antibody of thisdisclosure preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. For example, for the treatment of CXCR4⁺ tumors, a“therapeutically effective dosage” preferably inhibits cell growth ortumor growth by at least about 20%, more preferably by at least about40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects. Theability of a compound to inhibit tumor growth can be evaluated in ananimal model system predictive of efficacy in human tumors.Alternatively, this property of a composition can be evaluated byexamining the ability of the compound to inhibit cell growth, suchinhibition can be measured in vitro by assays known to the skilledpractitioner. A therapeutically effective amount of a therapeuticcompound can decrease tumor size, or otherwise ameliorate symptoms in asubject. One of ordinary skill in the art would be able to determinesuch amounts based on such factors as the subject's size, the severityof the subject's symptoms, and the particular composition or route ofadministration selected.

A composition of the present disclosure can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for antibodies of thisdisclosure include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, an antibody of this disclosure can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of this disclosure can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent disclosure include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of thisdisclosure can be formulated to ensure proper distribution in vivo. Forexample, the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of this disclosurecross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

Uses and Methods

The antibodies, particularly the human antibodies, antibody compositionsand methods of the present disclosure have numerous in vitro and in vivodiagnostic and therapeutic utilities involving the diagnosis andtreatment of CXCR4 mediated disorders. For example, these molecules canbe administered to cells in culture, in vitro or ex vivo, or to humansubjects, e.g., in vivo, to treat, prevent and to diagnose a variety ofdisorders. As used herein, the term “subject” is intended to includehuman and non-human animals. Non-human animals include all vertebrates,e.g., mammals and non-mammals, such as non-human primates, sheep, dogs,cats, cows, horses, chickens, amphibians, and reptiles. Preferredsubjects include human patients having disorders mediated by ormodulated by CXCR4 activity or involving the CXCR4/SDF-1 pathway. Whenantibodies to CXCR4 are administered together with another agent, thetwo can be administered in either order or simultaneously.

Given the specific binding of the antibodies of this disclosure forCXCR4, the antibodies of this disclosure can be used to specificallydetect CXCR4 expression on the surface of cells and, moreover, can beused to purify CXCR4 via immunoaffinity purification.

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multispecific and bispecific molecules andimmunoconjugates) of this disclosure in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

As previously described, human anti-CXCR4 antibodies of this disclosurecan be co-administered with one or other more therapeutic agents, e.g.,a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Theantibody can be linked to the agent (as an immunocomplex) or can beadministered separate from the agent. In the latter case (separateadministration), the antibody can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, and cyclophosphamide hydroxyurea which, by themselves, areonly effective at levels which are toxic or subtoxic to a patient.Cisplatin is intravenously administered as a 100 mg/kg dose once everyfour weeks and adriamycin is intravenously administered as a 60-75 mg/mldose once every 21 days. Co-administration of the human anti-CXCR4antibodies, or antigen binding fragments thereof, of the presentdisclosure with chemotherapeutic agents provides two anti-cancer agentswhich operate via different mechanisms which yield a cytotoxic effect tohuman tumor cells. Such co-administration can solve problems due todevelopment of resistance to drugs or a change in the antigenicity ofthe tumor cells that would render them unreactive with the antibody.

Target-specific effector cells, e.g., effector cells linked tocompositions (e.g., human antibodies, multispecific and bispecificmolecules) of this disclosure can also be used as therapeutic agents.Effector cells for targeting can be human leukocytes such asmacrophages, neutrophils or monocytes. Other cells include eosinophils,natural killer cells and other IgG- or IgA-receptor bearing cells. Ifdesired, effector cells can be obtained from the subject to be treated.The target-specific effector cells can be administered as a suspensionof cells in a physiologically acceptable solution. The number of cellsadministered can be in the order of 10⁸-10⁹ but will vary depending onthe therapeutic purpose. In general, the amount will be sufficient toobtain localization at the target cell, e.g., a tumor cell expressingCXCR4, and to effect cell killing by, e.g., phagocytosis. Routes ofadministration can also vary.

Therapy with target-specific effector cells can be performed inconjunction with other techniques for removal of targeted cells. Forexample, anti-tumor therapy using the compositions (e.g., humanantibodies, multispecific and bispecific molecules) of this disclosureand/or effector cells armed with these compositions can be used inconjunction with chemotherapy. Additionally, combination immunotherapymay be used to direct two distinct cytotoxic effector populations towardtumor cell rejection. For example, anti-CXCR4 antibodies linked toanti-Fc-gamma RI or anti-CD3 may be used in conjunction with IgG- orIgA-receptor specific binding agents.

Bispecific and multispecific molecules of this disclosure can also beused to modulate FcγR or FcγR levels on effector cells, such as bycapping and elimination of receptors on the cell surface. Mixtures ofanti-Fc receptors can also be used for this purpose.

The compositions (e.g., human, humanized, or chimeric antibodies,multispecific and bispecific molecules and immunoconjugates) of thisdisclosure which have complement binding sites, such as portions fromIgG1, -2, or -3 or IgM which bind complement, can also be used in thepresence of complement. In one embodiment, ex vivo treatment of apopulation of cells comprising target cells with a binding agent of thisdisclosure and appropriate effector cells can be supplemented by theaddition of complement or serum containing complement. Phagocytosis oftarget cells coated with a binding agent of this disclosure can beimproved by binding of complement proteins. In another embodiment targetcells coated with the compositions (e.g., human antibodies,multispecific and bispecific molecules) of this disclosure can also belysed by complement. In yet another embodiment, the compositions of thisdisclosure do not activate complement.

The compositions (e.g., human, humanized, or chimeric antibodies,multispecific and bispecific molecules and immunoconjugates) of thisdisclosure can also be administered together with complement.Accordingly, within the scope of this disclosure are compositionscomprising human antibodies, multispecific or bispecific molecules andserum or complement. These compositions are advantageous in that thecomplement is located in close proximity to the human antibodies,multispecific or bispecific molecules. Alternatively, the humanantibodies, multispecific or bispecific molecules of this disclosure andthe complement or serum can be administered separately.

The antibodies of this disclosure also can be used in combination withone or more additional therapeutic antibodies or other binding agents,such as Ig fusion proteins. Non-limiting examples of other antibodies orbinding agents with which an anti-CXCR4 antibody of this disclosure canbe administered in combination include antibodies or binding agents toCTLA-4, PSMA, CD30, IP-10, IFN-γ, CD70, PD-1, PD-L1, TNF, TNF-R, VEGF,VEGF-R, CCR5, IL-1, IL-18, IL-18R, CD19, Campath-1, EGFR, CD33, CD20,Her-2, CD25, gpIIb/IIIa, IgE, CD11a, α4 integrin, IFNα and IFNAR1.

Also within the scope of the present disclosure are kits comprising theantibody compositions of this disclosure (e.g., human antibodies,bispecific or multispecific molecules, or immunoconjugates) andinstructions for use. The kit can further contain one or more additionalreagents, such as an immunosuppressive reagent, a cytotoxic agent or aradiotoxic agent, or one or more additional human antibodies of thisdisclosure (e.g., a human antibody having a complementary activity whichbinds to an epitope in the CXCR4 antigen distinct from the first humanantibody).

Accordingly, patients treated with antibody compositions of thisdisclosure can be additionally administered (prior to, simultaneouslywith, or following administration of a human antibody of thisdisclosure) with another therapeutic agent, such as a cytotoxic orradiotoxic agent, which enhances or augments the therapeutic effect ofthe human antibodies.

In other embodiments, the subject can be additionally treated with anagent that modulates, e.g., enhances or inhibits, the expression oractivity of Fcγ or Fcγ receptors by, for example, treating the subjectwith a cytokine. Preferred cytokines for administration during treatmentwith the multispecific molecule include of granulocytecolony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumornecrosis factor (TNF).

The compositions (e.g., human antibodies, multispecific and bispecificmolecules) of this disclosure can also be used to target cellsexpressing CXCR4, for example for labeling such cells. For such use, thebinding agent can be linked to a molecule that can be detected. Thus,this disclosure provides methods for localizing ex vivo or in vitrocells expressing CXCR4. The detectable label can be, e.g., aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.

In a particular embodiment, this disclosure provides methods fordetecting the presence of CXCR4 antigen in a sample, or measuring theamount of CXCR4 antigen, comprising contacting the sample, and a controlsample, with a human monoclonal antibody, or an antigen binding portionthereof, which specifically binds to CXCR4, under conditions that allowfor formation of a complex between the antibody or portion thereof andCXCR4. The formation of a complex is then detected, wherein a differencecomplex formation between the sample compared to the control sample isindicative the presence of CXCR4 antigen in the sample.

In yet another embodiment, immunoconjugates of this disclosure can beused to target compounds (e.g., therapeutic agents, labels, cytotoxins,radiotoxoins immunosuppressants, etc.) to cells which have CXCR4 cellsurface receptors by linking such compounds to the antibody. Thus, thisdisclosure also provides methods for localizing ex vivo or in vivo cellsexpressing CXCR4 (e.g., with a detectable label, such as a radioisotope,a fluorescent compound, an enzyme, or an enzyme co-factor).Alternatively, the immunoconjugates can be used to kill cells which haveCXCR4 cell surface receptors by targeting cytotoxins or radiotoxins toCXCR4.

CXCR4 is known to be expressed on a wide variety of tumor cells typesand also is known to be involved in tumor metastasis. Moreover, as acoreceptor for HIV entry into T cells, CXCR4 is known to be involved inHIV infection. Additionally, the CXCR4/SDF-1 pathway has been shown tobe involved in inflammatory conditions. Still further, the CXCR4/SDF-1pathway has been shown to be involved in angiogenesis orneovascularization. Accordingly, the anti-CXCR4 antibodies (andimmunoconjugates and bispecific molecules) of this disclosure can beused to modulate CXCR4 activity in each of these clinical situations, asfollows:

A. Cancer

CXCR4 has been shown to be expressed by a variety of cancer types and incertain situations an inverse correlation has been established betweenCXCR4 expression and patient prognosis or survival. Non-limitingexamples of cancer types associated with CXCR4 expression include:breast (Muller, A. et al. (2001) Nature 410:50-56); ovarian (Scotton, C.et al. (2001) Br. J. Cancer 85:891-897; prostate (Taichman, R. S. et al.(2002) Cancer Res. 62:1832-1837; non-small cell lung (Spano J. P. et al.(2004) Ann. Oncol. 15:613-617); pancreatic (Koshiba, T. et al. (2000)Clin. Cancer Res. 6:3530-3535); thyroid (Hwang, J. H. et al. (2003) J.Clin. Endocrinol. Metab. 88:408-416); nasopharyngeal carcinoma (Wang, N.et al. (2005) Transl. Med. 3:26-33); melanoma (Scala, S. et al. (2005)Clin. Cancer Res. 11:1835-1841); renal cell carcinoma (Staller, P. etal. (2003) Nature 425:307-311); lymphoma (Bertolini, F. et al. (2002)Cancer Res. 62:3530-3535); neuroblastoma (Geminder, H. et al. (2001) J.Immunol. 167:4747-4757); glioblastoma (Rempel, S. A. et al. (2000) Clin.Cancer Res. 6:102-111); rhabdomyosarcoma (Libura, J. et al. (2002) Blood100:2597-2606); colorectal (Zeelenberg, I. S. et al. (2003) Cancer Res.63:3833-3839); kidney (Schrader, A. J. et al. (2002) Br. J. Cancer86:1250-1256); osteosarcoma (Layerdiere, C. et al. (2005) Clin. CancerRes. 11:2561-2567); acute lymphoblastic leukemia (Crazzolara, R. et al.(2001) Br. J. Haematol. 115:545-553); and acute myeloid leukemia(Rombouts, E. J. C. et al. (2004) Blood 104:550-557).

In view of the foregoing, the anti-CXCR4 antibodies of this disclosurecan be used in the treatment of cancers, including but not limited to acancer selected from the group consisting of breast, ovarian, prostate,non-small cell lung, pancreatic, thyroid, nasopharyngeal carcinoma,melanoma, renal cell carcinoma, lymphoma, neuroblastoma, glioblastoma,rhabdomyosarcoma, colorectal, kidney, osteosarcoma, acute lymphoblasticleukemia and acute myeloid leukemia. The antibody can be used alone orin combination other cancer treatments, such as surgery and/orradiation, and/or with other anti-neoplastic agents, such as theanti-neoplastic agents discussed and set forth above, includingchemotherapeutic drugs and other anti-tumor antigen antibodies, such asthose that bind CD20, Her2, PSMA, Campath-1, EGFR and the like.

B. Viral Infections, Including HIV Infection

CXCR4 has been shown to be a coreceptor for HIV entry into T cells and,additionally, certain murine anti-CXCR4 antibodies have beendemonstrated to be able to inhibit entry of HIV isolates into T cells(see Hou, T. et al. (1998) J. Immunol. 160:180-188; Carnec, X. et al.(2005) J. Virol. 79:1930-1938). Thus, CXCR4 can be used as a receptor byviruses for entry into the cell and antibodies to CXCR4 can be used toinhibit cell entry of such viruses that use CXCR4 as a receptor.Accordingly, the human anti-CXCR4 antibodies of this disclosure can beused to inhibit entry of a virus into a cell, wherein the virus usesCXCR4 as a receptor for cell entry, such that viral infection isinhibited. In a preferred embodiment, the antibodies are used to inhibitentry of HIV into T cells, e.g., in the treatment or prevention ofHIV/AIDS. The antibody can be used alone or in combination with otheranti-viral agents, such as anti-retroviral drugs such as AZT or proteaseinhibitors.

C. Inflammatory Conditions

The CXCR4/SDF-1 pathway has been shown to play a role in a variety ofinflammatory conditions, including but not limited to inflammatory liverdisease (Terada, R. et al. (2003) Lab. Invest. 83:665-672); autoimmunejoint inflammation (Matthys, P. et al. (2001) J. Immunol.167:4686-4692); allergic airway disease (Gonzalo, J. A. et al. (2000) J.Immunol. 165:499-508); and periodontal disease (Hosokawa, Y. et al.(2005) Clin. Exp. Immunol. 141:467-474).

Accordingly, the human anti-CXCR4 antibodies of this disclosure thatinhibit binding of SDF-1 to CXCR4 can be used to inhibit inflammation ininflammatory disorders, including disorders selected from the groupconsisting of inflammatory liver disease, autoimmune joint inflammation,allergic airway disease, periodontal disease, rheumatoid arthritis,inflammatory bowel disease, systemic lupus erythematosus, Type Idiabetes, inflammatory skin disorders (e.g., psoriasis, lichen planus),autoimmune thyroid disease, Sjogren's syndrome, pulmonary inflammation(e.g., chronic obstructive pulmonary disease, pulmonary sarcoidosis,lymphocytic alveolitis) and inflammatory kidney disease (e.g., IgAnephropathy, glomerulonephritis). The antibody can be used alone or incombination with other anti-inflammatory agents, such as non-steroidalanti-inflammatory drugs (NSAIDs), corticosteroids (e.g., prednisone,hydrocortisone), methotrexate, COX-2 inhibitors, TNF antagonists (e.g.,etanercept, infliximab, adalimumab) and immunosuppressants (such as6-mercaptopurine, azathioprine and cyclosporine A).

D. Angiogenesis

It has been demonstrated that SDF-1 induces neovascularization throughrecruitment of CXCR4-expressing hemangiocytes (Jin, D. K. et al. (2006)Nat. Med. 12:557-567). Moreover, blockade of the SDF-1/CXCR4 pathway canattenuate in vivo tumor growth by inhibiting angiogenesis in aVEGF-independent manner (Guleng, B. et al. (2005) Cancer Res.65:5864-58-71). Still further, as demonstrated in Example 2, antibodiesof this disclosure are capable of inhibiting capillary tube formation invitro. Accordingly, the anti-CXCR4 antibodies of this disclosure thatinhibit binding of SDF-1 to CXCR4 can be used to inhibit angiogenesis byinterfering with the SDF-1/CXCR4 pathway. Inhibition of angiogenesis canbe used, for example, to inhibit tumor growth or tumor metastasis(regardless of whether the tumor is CXCR4⁺). The antibody can be usedalone or in combination with other anti-angiogenic agents, such asanti-VEGF antibodies.

E. Autologous Stem Cell Transplantation

Peripheral blood stem cells are the preferred source of stem cells foruse in autologous stem cell transplantation, for example in thetreatment of certain hematological malignancies. Collection of stemcells from the peripheral blood requires mobilization of CD34⁺ stemcells from the bone marrow to the peripheral blood. Various cytokines,chemokines and adhesion molecules have been implicated in the regulationof this process (reviewed in Gazitt, Y. (2001) J. Hematother. Stem CellRes. 10:229-236), including the interaction of CXCR4 and SDF-1.Moreover, a small molecule CXCR4 antagonist has been demonstrated tostimulate rapid mobilization of CD34⁺ stem cells from the bone marrow tothe periphery (see e.g., Devine, S. M. et al. (2004) J. Clin. Oncol.22:1095-1102; Broxmeyer, H. E. et al. (2005) J. Exp. Med. 201:1307-1318;Flomenberg, N. et al. (2005) Blood 106:1867-1874). Accordingly,anti-CXCR4 antibodies of this disclosure that inhibit CXCR4 activity(i.e., antagonist antibodies) can be used to stimulate mobilization ofCD34⁺ stem cells from the bone marrow to the peripheral blood to allowfor the use of such stem cells in transplantation (e.g., autologoustransplantation), for example in the treatment of hematologicaldisorders, such as multiple myeloma and non-Hodgkin's lymphoma. Theantibody can be used alone or in combination with other agents used tostimulate mobilization of stem cells, such as G-CSF and/or GM-CSF. Thus,in another embodiment, the invention provides a method of stimulatingmobilization of CD34⁺ stem cells from bone marrow to peripheral blood ina subject, the method comprising administering to the subject ananti-CXCR4 antibody of the invention such that mobilization of CD34⁺stem cells from bone marrow to peripheral blood is stimulated. Themethod can further comprise collecting CD34+ stem cells from peripheralblood, such as for use in autologous stem cell transplantation.

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Example 1 Generation of Human Monoclonal Antibodies AgainstCXCR4

Anti-CXCR4 human monoclonal antibodies were generated using acombination approach in which, first, mice expressing human antibodygenes were immunized to raise in the mice a repertoire of humanimmunoglobulins specific for human CXCR4 and then, second, a humanantibody library was prepared from spleen cells of the mice anddisplayed on phage such that the phage were then screened for expressionof antibodies with specificity for CXCR4. This combination approach isgenerally described in U.S. Application No. 20030091995 by Buechler etal.

Antigen

R1610 cells (a Chinese Hamster lung cell line, originally described inThirion, J. P. et al. (1976) Genetics 83:137-147) were transfected withan expression vector encoding the full-length human CXCR4 protein suchthat the protein was expressed on the surface of the cells. Acodon-optimized form of the CXCR4 cDNA was used in the expressionvector, which was prepared as described in Mirzabekov, T. et al. (1999)J. Biol. Chem. 274:28745-28750. To enhance the immunogenicity of thecells, the cells were coated with trinitrophenol (TNP), by incubationwith an aqueous solution of trinitrobenzenesulfonic acid (TNBS),available commercially as a 5% solution (Sigma, Cat. #P2297). Morespecifically, 1×10⁸ cells were washed once with sterile PBS, incubatedwith 50 μl of the commercial 5% TNBS solution for one hour in the darkat room temperature and then washed three times with PBS. The resultantTNP-coated, CXCR-4-expressing R1610 cells were used as antigen forimmunization. The final immunogen was a mix of 100 μl of TNP-coated,washed cells (1×10⁷ cells) plus 100 μl of Ribi adjuvant. Mice receivedsix doses of the immunogen over time.

Transgenic Transchromosomic KM Mouse® Strain

Fully human monoclonal antibodies to CXCR4 were prepared by initiallyimmunizing the KM strain of transgenic transchromosomic mice, whichexpresses human antibody genes. In this mouse strain, the endogenousmouse kappa light chain gene has been homozygously disrupted asdescribed in Chen et al. (1993) EMBO J. 12:811-820 and the endogenousmouse heavy chain gene has been homozygously disrupted as described inExample 1 of PCT Publication WO 01/09187. Additoinally, this mousestrain carries a human kappa light chain transgene, KCo5 (as describedin Fishwild et al. (1996) Nature Biotechnology 14:845-851) and alsocontains the SC20 transchromosome, which carries the human Ig heavychain locus, as described in PCT Publication WO 02/43478. KM mice arealso described in detail in U.S. Application No. 20020199213.

KM Immunization

To raise fully human monoclonal antibodies to CXCR4, mice of the KMMouse® strain were immunized with R1610 cells transfected to expressCXCR4 and coated with TNP (as described above for the antigen). Generalimmunization schemes for the raising human antibodies in mice strainscarrying human antibody genes are described in Lonberg, N. et al. (1994)Nature 368(6474): 856-859; Fishwild, D. et al. (1996) NatureBiotechnology 14: 845-851 and PCT Publication WO 98/24884. The mice were6-16 weeks of age upon the first infusion of antigen.

KM mice were immunized with antigen in Ribi adjuvant eitherintraperitonealy (IP), subcutaneously (Sc) or via footpad (FP), followedby 3-21 days IP, Sc or FP reimmunization (for a total of 6immunizations) with the antigen in Ribi adjuvant. The immune responsewas monitored by retroorbital bleeds. The plasma was screened by FACSstaining of CXCR4-expressing R1610 cells (versus untransfected parentalR1610 cells). Mice with sufficient titers of anti-CXCR4 humanimmunoglobulin were used for harvesting spleens.

Preparation of Phage Display Library and Screening forAnti-CXCR4Antibodies

Spleens harvested from the immunized mice described above were used tomake a phage display library expressing human antibody heavy and lightchains. More specifically total RNA was isolated from the spleens, cDNAwas prepared from the RNA and human antibody variable region cDNA wasspecifically amplified by PCR, essentially as described in U.S. PatentApplication 20030091995 by Buechler et al. The library of human antibodyvariable regions was cloned into phage expression vectors, againessentially as described in U.S. Patent Application 20030091995 byBuechler et al. The phage display library was screened for librarymembers having affinity for CXCR4 by panning with human CXCR4incorporated into magnetic proteoliposomes (CXCR4-MPL). MPLs expressingCXCR4, or other seven transmembrane (7TM) receptors (e.g., CCR5), suchthat the native conformation of the 7™ receptor is maintained, have beendescribed previously (see e.g., Mirzabekov, T. et al. (2000) Nat.Biotechnol. 18:649-654; Babcock, G. J. et al. (2001) J. Biol. Chem.276:38433-38440; PCT Publication WO 01/49265; U.S. Patent Application20010034432). In brief, recombinant human CXCR4 that contained anepitope tag was solublized from a transfected CXCR4-expressing cell lineusing the detergent CHAPSO and the protein was captured on magneticbeads via the epitope tag. A lipid membrane was reconstituted duringremoval of the detergent, such that the native membrane conformation ofCXCR4 was maintained, to create the CXCR4-MPLs. Three rounds of panningof the phage display library on the CXCR4-MPLs led to a 30-foldenrichment of CXCR4-binders as compared to background. Variable regionfragments of interest were recloned into a Fab expression vector and theFab retested for antigen binding against transfected CXCR4-expressingcells. Whole antibodies were then generated from the Fabs using standardmolecular biology techniques.

Fab clones F7, F9, D1 and E2 were selected for further analysis.

Example 2 Structural Characterization of Human Anti-CXCR4 MonoclonalAntibodies F7, F9, D1 and E2

The cDNA sequences encoding the heavy and light chain variable regionsof the F7, F9, D1 and E2 Fab clones, obtained from phage display libraryscreening as described in Example 1, were sequenced using standard DNAsequencing techniques.

The nucleotide and amino acid sequences of the heavy chain variableregion of F7 are shown in FIG. 1A and in SEQ ID NO: 33 and 25,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of F7 are shown in FIG. 1B and in SEQ ID NO: 37 and 29,respectively.

Comparison of the F7 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe F7 heavy chain utilizes a V_(H) segment from human germline V_(H)3-48, a D segment from the human germline 4-23, and a JH segment fromhuman germline JH 6B. Further analysis of the F7 V_(H) sequence usingthe Kabat system of CDR region determination led to the delineation ofthe heavy chain CDR1, CDR2 and CD3 regions as shown in FIG. 1A and inSEQ ID NOs: 1, 5 and 9, respectively.

Comparison of the F7 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe F7 light chain utilizes a V_(L) segment from human germline V_(K)L15 and a JK segment from human germline JK 1. Further analysis of theF7 V₁, sequence using the Kabat system of CDR region determination ledto the delineation of the light chain CDR1, CDR2 and CD3 regions asshown in FIG. 1B and in SEQ ID NOs: 13, 17 and 21, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of F9 are shown in FIG. 2A and in SEQ ID NO: 34 and 26,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of F9 are shown in FIG. 2B and in SEQ ID NO: 38 and 30,respectively.

Comparison of the F9 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe F9 heavy chain utilizes a V_(H) segment from human germline V_(H)3-48, a D segment from the human germline 4-23, and a JH segment fromhuman germline JR 6B. Further analysis of the F9 V_(H) sequence usingthe Kabat system of CDR region determination led to the delineation ofthe heavy chain CDR1, CDR2 and CD3 regions as shown in FIG. 2A and inSEQ ID NOs: 2, 6 and 10, respectively.

Comparison of the F9 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe F9 light chain utilizes a V_(L) segment from human germline V_(K)L15 and a JK segment from human germline JK 1. Further analysis of theF9 V_(L) sequence using the Kabat system of CDR region determination ledto the delineation of the light chain CDR1, CDR2 and CD3 regions asshown in FIG. 2B and in SEQ ID NOs: 14, 18 and 22, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of D1 are shown in FIG. 3A and in SEQ ID NO: 35 and 27,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of D1 are shown in FIG. 3B and in SEQ ID NO: 39 and 31,respectively.

Comparison of the D1 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe D1 heavy chain utilizes a V_(H) segment from human germline V_(H)3-48, a D segment from the human germline 4-23, and a JH segment fromhuman germline JH 6B. Further analysis of the D1 V_(H) sequence usingthe Kabat system of CDR region determination led to the delineation ofthe heavy chain CDR1, CDR2 and CD3 regions as shown in FIG. 3A and inSEQ ID NOs: 3, 7 and 11, respectively.

Comparison of the D1 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe D1 light chain utilizes a V_(L) segment from human germline V_(K)L15 and a JK segment from human germline JK 1. Further analysis of theD1 V_(L) sequence using the Kabat system of CDR region determination ledto the delineation of the light chain CDR1, CDR2 and CD3 regions asshown in FIG. 3B and in SEQ ID NOs: 15, 19 and 23, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of E2 are shown in FIG. 4A and in SEQ ID NO: 36 and 28,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of E2 are shown in FIG. 4B and in SEQ ID NO: 40 and 32,respectively.

Comparison of the E2 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe E2 heavy chain utilizes a V_(H) segment from human germline V_(H)3-48, a D segment from the human germline 4-23, and a JH segment fromhuman germline JH 6B. Further analysis of the E2 V_(H) sequence usingthe Kabat system of CDR region determination led to the delineation ofthe heavy chain CDR1, CDR2 and CD3 regions as shown in FIG. 4A and inSEQ ID NOs: 4, 8 and 12, respectively.

Comparison of the E2 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe E2 light chain utilizes a V_(L) segment from human germline V_(K)L15 and a JK segment from human germline JK 1. Further analysis of theE2 V_(L) sequence using the Kabat system of CDR region determination ledto the delineation of the light chain CDR1, CDR2 and CD3 regions asshown in FIG. 4B and in SEQ ID NOs: 16, 20 and 24, respectively.

Analysis of the framework sequences of the V_(H) and V_(L) regions ofF7, F9, D1 and E2, as compared to the germline sequences from which theywere derived, identified various framework amino acid residues thatdiffered from germline. Certain framework residues in the N-terminalregions of the V_(H) and V_(L) segments were chosen for “back-mutation”to restore the framework residue to the germline sequence, because thesenon-germline residues in the N-terminal portion were encoded by theprimers used to create the phage display libraries described inExample 1. In particular, the following modified forms of the V_(H) andV_(L) segments of F7, F9, D1 and E2 (referred to as “GL” forms, forgermline) were created using standard molecular biology techniques tosubstitute the germline amino acid residue at the indicated frameworkposition:

-   -   F7GL V_(H): Q1E, Q6E    -   F7GL V_(k): A1D, R3Q    -   F9GL V_(H): Q1E, Q6E    -   F9GL V_(k): E1D, V3Q, L4M    -   D1GL V_(H): Q6E    -   D1GL V_(k): V1D, W3Q, V4M    -   E2GL V_(H): Q6E    -   E2GL V_(k): E1D, V3Q, L4M

FIG. 5A shows the alignment of the F7 (SEQ ID NO: 25) and F7GL (SEQ IDNO: 41) heavy chain variable amino acid sequences with the germlineV_(H) 3-48 encoded amino acid sequence (SEQ ID NO: 49). The CDR1, CDR2and CDR3 regions are delineated.

FIG. 5B shows the alignment of the F7 (SEQ ID NO: 29) and F7GL (SEQ IDNO: 45) light chain variable amino acid sequences with the germlineV_(K) L15 encoded amino acid sequence (SEQ ID NO: 50). The CDR1, CDR2and CDR3 regions are delineated.

FIG. 6A shows the alignment of the F9 (SEQ ID NO: 26) and F9GL (SEQ IDNO: 42) heavy chain variable amino acid sequences with the germlineV_(H) 3-48 encoded amino acid sequence (SEQ ID NO: 49). The CDR1, CDR2and CDR3 regions are delineated.

FIG. 6B shows the alignment of the F9 (SEQ ID NO: 30) and F9GL (SEQ IDNO: 46) light chain variable amino acid sequences with the germlineV_(K) L15 encoded amino acid sequence (SEQ ID NO: 50). The CDR1, CDR2and CDR3 regions are delineated.

FIG. 7A shows the alignment of the D1 (SEQ ID NO: 27) and D1GL (SEQ IDNO: 43) heavy chain variable amino acid sequences with the germlineV_(H) 3-48 encoded amino acid sequence (SEQ ID NO: 49). The CDR1, CDR2and CDR3 regions are delineated.

FIG. 7B shows the alignment of the D1 (SEQ ID NO: 31) and D1GL (SEQ IDNO: 47) light chain variable amino acid sequences with the germlineV_(K) L15 encoded amino acid sequence (SEQ ID NO: 50). The CDR1, CDR2and CDR3 regions are delineated.

FIG. 8A shows the alignment of the E2 (SEQ ID NO: 28) and E2GL (SEQ IDNO: 44) heavy chain variable amino acid sequences with the germlineV_(H) 3-48 encoded amino acid sequence (SEQ ID NO: 49). The CDR1, CDR2and CDR3 regions are delineated.

FIG. 8B shows the alignment of the E2 (SEQ ID NO: 32) and E2GL (SEQ IDNO: 48) light chain variable amino acid sequences with the germlineV_(K) L15 encoded amino acid sequence (SEQ ID NO: 50). The CDR1, CDR2and CDR3 regions are delineated.

The F7, F9, D1 and E2 Fab fragments are converted to full-lengthantibodies using standard recombinant DNA techniques. For example, DNAencoding the V_(H) and V_(K) regions of one of the Fab fragments can becloned into an expression vector that carries the heavy and light chainconstant regions such that the variable regions are operatively linkedto the constant regions. Alternatively, separate vectors can be used forexpression of the full-length heavy chain and the full-length lightchain. Non-limiting examples of expression vectors suitable for use increating full-length antibodies include the pIE vectors described inU.S. Patent Application No. 20050153394 by Black.

Example 3 Binding Characteristics of Anti-CXCR4Human MonoclonalAntibodies

In this example, binding characteristics of the anti-CXCR4 antibodieswere examined by flow cytometry.

The human T cell line CEM, which expresses native human CXCR4 on itscell surface, was used to examine the ability of the F7, F9, D1 and E2antibodies to bind to native, cell-surface CXCR4. Full-length F7, F9, D1and E2 were titrated in a 1:3 serial dilution series, resulting in aconcentration range from 300 nM to 5 pM. The antibodies were then mixedwith CEM cells and allowed to bind before being detected with aFITC-conjugated anti-human IgG secondary antibody. The cells were thenanalyzed by fluorescent cytometry. The resulting mean fluorescenceintensities are shown in the graph of FIG. 9, which demonstrates thatall four anti-CXCR4 antibodies bind to CEM cells. The EC₅₀ for bindingF7, F9, D1 and E2 were 21 nM, 14 nM, 80 nM and 290 nM, respectively.

To determine the ability of a panel of anti-CXCR4 antibodies to competefor binding to CXCR4, competition studies were performed. The four humananti-CXCR4 antibodies F9, F7, E2 and D1 were used, along with fourcommercially available murine monoclonal anti-CXCR4 antibodies (12G5,708, 716 and 717; R&D Systems catalog #s: MAB170, MAB171, MAB172 andMAB173, respectively). The anti-CXCR4 antibodies were titrated in a 1:3serial dilution series resulting in a concentration range from 300 nM to5 pM in the presence of a constant concentration of FITC-labeledanti-CXCR4 antibody F9. The mixture of antibodies was then added to CEMcells and allowed to bind. The ability of each antibody to compete withF9 for binding to CEM cells was assessed by fluorescent cytometry anddetection of FITC. The resulting mean fluorescent intensitities areshown in the graph of FIG. 10, which demonstrates that all sevenantibodies examined (F7, E2, D1, 12G5, 708, 716 and 717) were able tocompete with F9 for binding to CEM cells, although the E2 antibody onlydemonstrated partial inhibition at high concentrations compared to theother antibodies.

In another set of experiments, the ability of the F7 mAb to bind to avariety of different cell lines was examined by flow cytometry bycarrying out a FACS titration. Increasing amounts of mAb (from less than0.001 μg/ml to more than 100 μg/ml) were incubated with 100,00 cells andbinding assessed by flow cytometry. The Bmax value also was determined,which indicates approximately how many CXCR4 molecules are present oneach cell. Based on the binding curves, an EC₅₀ for antibody binding wasdetermined, the results of which are summarized below in Table 1:

TABLE 1 FACS Titration Results for mAb F7 Binding to Different CellLines Cell Type EC₅₀ (μg/ml) Bmax Ramos 0.48 106,000 Raji 0.34 52,536Namalwa 1.57 116,000 L540 3.69 31,868 DMS79 3.99 24,587 MDA-MB-231 9.2414,186 Bmax = maximium binding (GMFI units)The results show that F7 mAb is capable of binding effectively to eachof the six cell lines tested, with the lowest EC₅₀s observed with theRamos and Raji cell lines. These data also show that the expression ofCXCR4 receptor is highest for Ramos and Namalwa cells and lowest forMDA-MB-231 cells and DMS79 cells.

In another binding experiment, the ability of the F7 mAb to bind todifferent subsets of human peripheral blood mononuclear cells (PBMCs)was examined. Human PBMCs were isolated by standard methods anddifferent cellular subsets were isolated by FACS. In particular, thefollowing cellular subsets were isolated: (i) CD3⁺, (ii) CD20⁺; (iii)CD11b⁺ and (iv) CD14⁺. Flow cytometry experiments conducted with the F7mAb (at 33 μg/ml) demonstrated that the F7 mAb was capable of bindingeffectively to each of the four subsets, as compared to anisotype-matched control antibody.

Example 4 Inhibition of SDF-1 Binding to CXCR4 by Anti-CXCR4Antibodies

To determine the ability of the anti-CXCR4 human antibodies to inhibitthe binding of SDF-1 to CXCR4, a competition study was performed using¹²⁵I-labeled SDF-1 and CEM cells, which naturally express CXCR4. Acomparison of anti-CXCR4 antibodies on blocking SDF-1 binding to CEMcells was performed by a standard radio-labeled ligand binding assay.The anti-CXCR4 antibodies were serially diluted 1:3 to yield a range ofconcentrations from 300 nM to 137 pM. The antibodies were added to750,000 CEM cells in 100 μl in the presence of 100 pM ¹²⁵I-SDF-1 with aspecific activity of 2000 Ci/mmole (Amersham, catalog #IM314-25UCI). Anirrelevant antibody of the same isotype was used as a negative control.The total possible bound radio-labeled ligand was determined by allowingthe ¹²⁵I-SDF-1 to bind to CEM cells in the absence of antibodies for 2hours at 4° C. Non-specific binding of the radio-labeled ligand wasdetermined by allowing the ¹²⁵I-SDF-1 to bind in the presence of 1 μMunlabeled SDF-1 (Peprotech, catalog #300-28A). The amount ofcell-associated ¹²⁵I-SDF-1 was determined by standard methods. Theresults are shown in FIG. 11, which demonstrates that the F7 antibodyprovides the most effective blockade of SDF-1 binding to CXCR4 expressedon CEM cells. The F9 and D1 antibodies also blocked SDF-1 binding,although more moderately than F7. The E2 antibody, although it does bindto CXCR4 on CEM cells (as demonstrated in Example 3), did noteffectively block SDF-1 binding to CXCR4 on CEM cells. The EC₅₀s forSDF-1 blockade by F7, F9 and D1 were 2.3 nM, 12.5 nM and 28.6 nM,respectively.

Example 5 Inhibition of SDF-1-Induced Calcium Flux by Anti-CXCR4Antibodies

To determine the ability of the anti-CXCR4 human antibodies to inhibitcalcium flux in CEM cells induced by SDF-1, CEM cells were first labeledwith the fluorescent dye Calcium 3 (Molecular Devices). The anti-CXCR4antibodies were titrated in a 1:3 serial dilution series resulting in aconcentration range from 100 nM to 1 μM and allowed to bind to 200,000CEM cells in 200 μl and incubated 10 minutes at room temperature priorto loading into a Flexstation machine (Molecular Devices). As a negativecontrol, an irrelevant antibody of the same isotype was used. Cells werethen stimulated with a final concentration of 50 nM recombinant humanSDF-1α (Peprotech), added as 500 nM in a volume of 22 μl for a finalvolume of 222 μl. The resulting calcium flux was measured for 200seconds per well. As a positive control, cells in the absence ofantibody were stimulated with SDF-1α (made up in Hank's buffered saline(HBS) with 0.1% BSA or HBS) to achieve a maximum possible calcium fluxsignal. To determine a baseline, cells were stimulated with HBS with0.1% BSA. The SDF-1α-stimulated release of calcium was measured by thedevelopment of calcium-dependent fluorescence over time. The area underthe curve of the resulting fluorescence trace was reported as anindication of calcium flux. The resulting inhibition of calcium flux bythe anti-CXCR4 antibodies is represented in FIG. 12. The data wereplotted and the EC₅₀s were calculated using GraphPad Prism software andthe non-linear curve fit, sigmoidal dose response formula. AntibodiesF7, F9 and D1 inhibited SDF-1α-induced calcium flux. Antibody E2,although it did bind to CXCR4 (as demonstrated in Example 3), did notsignificantly inhibit SDF-1α-induced calcium flux. The EC₅₀s forinhibition of SDF-1-induced calcium flux by F7, F9 and D1 were 0.90 nM,0.32 nM and 0.57 nM, respectively.

Example 6 Inhibition of SDF-1-Induced Migration of CEM Cells byAnti-CXCR4Antibodies

To determine the ability of the anti-CXCR4 human antibodies to inhibitmigration of CEM cells induced by SDF-1, CEM cells first were labeledwith the BATDA reagent (Perkin Elmer). The anti-CXCR4 antibodies weretitrated in a 1:3 serial dilution series resulting in a concentrationrange from 100 nM to 1 μM and allowed to bind to labeled CEM cells at adensity of 10 million cells per ml. As a negative control, an irrelevantantibody of the same isotype was used. Recombinant human SDF-1α(Peprotech) was added at 5 nM at 30 μl per well to the lower chamber ofa 96 well Neuroprobe migration plate with 5.7 mm diameter filters perwell. Each well contains 5 μM pores. Labeled CEM cells with and withoutantibody were loaded onto the filters at a concentration of 0.5 millioncells per well in a volume of 50 μl. The migration plate was incubatedat 37° C. for 2.5 hours. Migrated cells were captured in the lowerchamber of the plate, lysed and detected with Europium detectionsolution (Perkin Elmer). The chemi-luminescent signal was recorded on aFusion instrument. The resulting inhibition of SDF-1a-induced migrationby the anti-CXCR4 antibodies in shown in FIG. 13. The resultsdemonstrated that antibodies F7 and F9 inhibited migration effectively,while antibodies D1 and E2 did not significantly inhibit migration. TheEC₅₀s for inhibition of SDF-1-induced CEM cell migration by F7 and F9were 12.44 nM and 18.99 nM, respectively.

Example 7 Inhibition of HuVEC Capillary Tube Formation by Anti-CXCR4Antibodies

In this example, the ability of the anti-CXCR4 human antibodies toinhibit capillary tube formation by human umbilical vein endothelialcells (HuVEC) was examined. Matrigel was diluted 1:1 with RPMI andplated onto the wells of a 96 well plate and allowed to polymerize for30 minutes at 37° C. HuVEC (from Cambrex, cat. # CC-2519) at 80%confluence were trypsanized and resuspended at 1×10⁶ cells per ml inRPMI with 0.5% FBS. Antibodies were well mixed with HuVEC at a finalconcentration of 3 μg/ml and allowed to incubate at room temperature for30 minutes. An irrelevant antibody of the same isotype or media alonewas used as a negative control. As a positive control of inhibition oftube formation, a mouse anti-human αvβ3 (CD51/CD61) antibody (R&DSystems, cat. # MAB3050) was used. HuVEC with or without antibodies wereplated onto the matrigel-coated wells and incubated at 37° C. for 18hours.

The HuVEC incubated with media alone or with the isotype-matched controlantibody formed capillary tubes resulting in the appearance of connectedcells across the plate with 3-5 points of connection or branch pointsper cell. The HuVEC incubated with either the anti-CXCR4 humanantibodies or the anti-αvβ3 antibody did not form capillary tubes. Thecells appeared isolated and with few or no branch points. The anti-CXCR4antibodies that were most effective in blocking SDF-1 binding,SDF-1-induced calcium flux and SDF-1-induced migration, namely F7 andF9, were also the most effective in inhibiting capillary tube formation.The anti-CXCR4 antibody E2, which binds to CXCR4 but does not blockSDF-1 binding or SDF-1-induced effects, did not inhibit capillary tubeformation.

Example 8 Inhibition of Tumor Cell Proliferation In Vitro by Anti-CXCR4Antibodies

In this example, the ability of the anti-CXCR4 human antibodies toinhibit proliferation of Ramos tumor cells (a human Burkitt's lymphomacell line) in vitro was examined. In the assay, 1×10⁴ cells/well wereincubated with increasing doses (10⁻³ to 300 nM) of F7 IgG4 antibody, F9IgG1 antibody, E2 IgG1 antibody, F9 Fab′ antibody or isotype controls.The cells were incubated with antibody for 72 hours, with ³H-thymidinebeing added for the final 24 hours of incubation to allow for monitoringof cell proliferation. Following the incubation, incorporation of³H-thymidine by the cells was measured by standard techniques. Theresults are shown in the graph of FIG. 14. The results demonstrate thatthe F7 IgG4, F9 IgG1 and E2 IgG1 antibodies each were able to inhibitRamos cell proliferation, as indicated by decreased ³H-thymidineincorporation when incubated with these antibodies, whereas the F9 Fab′fragment did not inhibit cell proliferation. These results indicate thatthe anti-CXCR4 human antibodies have a direct anti-proliferative effecton the tumor cells in vitro and thus do not require secondarycross-linking to achieve an anti-proliferative effect.

Example 9 Inhibition of Solid Tumor Cell Proliferation In Vivo byAnti-CXCR4 Antibodies

In this example, the ability of the anti-CXCR4 human antibodies toinhibit proliferation of an established solid tumor in vivo was examinedusing a Ramos subcutaneous tumor cell model. In this assay, 10×10⁶ Ramoscells/mouse were implanted into the flank region of each mouse andallowed to grow to a mean size of 40 mm³, calculated bylength×width×height/2 of the tumors. The mice then received anintraperitoneal (i.p.) injection of a first dose of antibody (designatedas day 0 of treatment) and received a second i.p. dose of antibody onday 7. Mice treated with a Fab′ fragment antibody also received i.p.antibody doses on day 3 and day 10. Groups of mice (n=8) were treatedwith either (i) vehicle; (ii) isotype control (15 mg/kg); (iii) F7 IgG4(15 mg/kg); (iv) F9 IgG1 (15 mg/kg); (v) F9 Fab′ (10 mg/kg); or (vi)anti-CD20 positive control (15 mg/kg). Tumor volume and mouse bodyweight were measured at regular intervals (approximately 2-3 times/week)between day 0 and day 30 post dosing. The results of the experiment arepresented in FIGS. 15A, 15B and 15C, which show mean tumor volume (FIG.15A), median tumor volume (FIG. 15B) and median % body weight change(FIG. 15C). The results demonstrated that, like the positive control,the F7 IgG4 and F9 IgG1 antibodies significantly inhibited tumor cellgrowth as measured by increased tumor volume, whereas the F9 Fab′fragment did not inhibit tumor cell growth as compared to the isotypecontrol. All treatments were well-tolerated as indicated by nosignificant body weight change. The differences in body weights betweentreatments was most likely due to the weights of the tumors. The resultsindicate that the anti-CXCR4 human antibodies are capable of inhibitinggrowth of an established solid tumor in vivo.

Example 10 Increased Survival Time in a Mouse Systemic Tumor Cell Modelby Treatment with an Anti-CXCR4Antibody

In this example, the ability of an anti-CXCR4 human antibody to increasesurvival time of mice was examined using a Ramos systemic tumor cellmodel. In this assay, 1×10⁶ Ramos cells/mouse were injectedintravenously (i.v.) into each mouse on day 0. The mice then received anintraperitoneal (i.p.) injection of a first dose of antibody on day 1(i.e., one day after i.v. administration of tumor cells) and receivedfour more i.p. doses of antibody, on days 5, 8, 15 and 22 (mice treatedwith the positive control antibody were treated only on day 1). Groupsof mice (n=8) were treated with either (i) vehicle; (ii) isotype control(15 mg/kg); (iii) F9 IgG1 (15 mg/kg); or (iv) anti-CD19 positive control(15 mg/kg). Percent survival was measured at regular intervals betweenday 0 and day 50 post dosing (hind leg paralysis was used as theendpoint of the experiment). The results of the experiment are presentedin FIG. 16, which shows percent survival over time. The median # days ofsurvival for the mice treated with either vehicle or the isotype controlwere 23 and 25.5 days, respectively, whereas the median # days ofsurvival of the mice treated with one dose of the anti-CD19 positivecontrol was 39 days. Significantly, 100% of the mice in the grouptreated with five doses of the F9 IgG1 antibody survived to the end ofthe experiment. These results indicate that the anti-CXCR4 humanantibody is capable of increasing survival times of mice in a systemictumor cell model.

Example 11 Induction of Apoptosis by Anti-CXCR4Monoclonal Antibody F7

In this example, the ability of the anti-CXCR4 mAb F7 to induceapoptosis in different cells was examined. In the apoptosis assay, F7mAb at 10 μg/ml was incubated with either Ramos cells (500,000 cells),Namalwa cells (500,000 cells) or R1610 cells transfected to expressCXCR4 (100,000 cells) Untransfected R1610 cells were used as a negativecontrol. Anti-CXCR4 mAb F7 or isotype control antibody was incubatedwith cells at 37° C. and 250 μl samples were removed at 24, 48 and 72hours. To assess apoptosis, the cells from various time points wereincubated with Annexin V-FITC-FL1 and Propidium Iodide—FL3, followed byflow cytometry. The combined percentage of cells collected in the FL1,FL3 and FL1-FL3 double positive quadrants were considered apoptotic. Toremove background, the percentages of isotype antibody—induced apoptoticcells was subtracted from the percentage of F7 mAb-induced apoptoticcells.

The results are summarized below in Table 2:

TABLE 2 Induction of Apoptosis by Anti-CXCR4 mAb F7 Cells Time (Hours) %Apoptosis R1610 72 <1 R1610-CXCR4 24 39 R1610-CXCR4 48 58 R1610-CXCR4 7246 Ramos 24 22 Ramos 48 31 Ramos 72 22 Namalwa 24 17 Namalwa 48 24Namalwa 72 44 Total % apoptosis values are corrected for basleinechanges induced by isotype control antibodies.The results demonstrate that the F7 mAb is capable of inducing apoptosisin the Ramos, Namalwa and R1610-CXCR4 cells while F7 had no effect oninduction of apoptosis of parental R1610 cells indicating that theresponse was CXCR4-specific.

Example 12 Additional Studies Showing Inhibition of Solid Tumor CellProliferation

In Vivo by Anti-CXCR4Antibodies

In this example, the ability of anti-CXCR4 human antibodies to inhibitproliferation or induce apoptosis of established solid tumors in vivowas examined using additional tumor cell models similar to the Ramosmodel described above in Example 9. A variety of tumor cell lines wereexamined. Representative experiments and results are as follows.

In one experiment, 7.5×10⁶ MDA-MB231 human breast cancer cells/mousewere implanted into the flank region of each mouse and allowed to growto a mean size of 100 mm³, calculated by length×width×height/2 of thetumors, which was day 7 post-implantation. The mice were randomized intodifferent treatment groups and received an intraperitoneal (i.p.)injection of a first dose of antibody on day 7 post-implantation,received a second i.p. dose of antibody on day 14 post-implantation andthen received a third dose on day 46 post-implantation. Groups of mice(n=9) were treated with either (i) vehicle (PBS); (ii) IgG1 isotypecontrol (15 mg/kg); (iii) IgG4 isotype control (15 mg/kg); (iv) F7 IgG1(15 mg/kg); or (v) F7 IgG4 (15 mg/kg). Tumor volumes were measured atregular intervals and the mean and median tumor volume determined foreach treatment group at each interval. The results of this experimentare summarized below in Table 3, which shows mean tumor volume (in mm³)and % tumor growth inhibition (TGI) at day 52, and median tumor volume(in mm³) and % TGI at day 59 post-implantation:

TABLE 3 Tumor Growth Inhbition of MDA-MB231 Cells In Vivo by mAb F7 Day52 Day 59 Treatment Mean TGI (%) Median TGI (%) Vehicle 154 187 IgG1Isotype Control 172 216 IgG4 Isotype Control 188 226 F7 Anti-CXCR4 IgG186 50 130 40 F7 Anti-CXCR4 IgG4 79 58 108 52Additionally, one of the mice in the F7 IgG4 treatment group was tumorfree at day 59. The results demonstrate that the F7 mAb is capable ofinhibiting growth of MDA-MB231 breast cancer cells in vivo.

In a second experiment, 5×10⁶ DMS79 human small cell lung carcinomacells/mouse were implanted into the flank region of each mouse andallowed to grow to a mean size of 160 mm³, calculated bylength×width×height/2 of the tumors, which was day 7 post-implantation.The mice were randomized into different treatment groups and receivedintraperitoneal (i.p.) injections of antibody on a dosing schedule ofQ3Dx5 (every three days for five times). Groups of mice (n=10) weretreated with either (i) vehicle (PBS); (ii) IgG4 isotype control (10mg/kg); or (iii) F7 IgG4 (10 mg/kg). Tumor volumes were measured atregular intervals and the mean and median tumor volume determined foreach treatment group at each interval. The results of this experimentare summarized below in Table 4, which shows mean and median tumorvolume (in mm³) and % tumor growth inhibition (TGI) at day 34post-implantation:

TABLE 4 Tumor Growth Inhbition of DMS79 Cells In Vivo by mAb F7 Day 34Treatment Mean TGI (%) Median TGI (%) Vehicle 900 882 IgG4 IsotypeControl 992 903 F7 Anti-CXCR4 IgG4 620 38 599 34The results demonstrate that the F7 mAb is capable of inhibiting growthof DMS79 human small cell lung carcinoma cells in vivo.

Additional subcutaneous xenograft tumor models were tested for theability of anti-CXCR4 antibodies to inhibit tumor growth, in experimentssimilar to those described above and in Example 9. In an experimentusing SU-DHL-6 B cell lymphoma cells, the results showed that treatmentwith the F7 IgG4 mAb at 15 mg/kg resulted in approximately 60% tumorgrowth inhibition. Similarly, in an experiment using Namalwa Burkitt'slymphoma cells, the results showed that treatment with the F7 IgG4 mAbat 3 mg/kg resulted in approximately 70% tumor growth inhibition. Incontrast, no tumor growth inhibition by the F7 mAb was observed inexperiments using NIH-H226 lung carcinoma cells or HPAC human pancreaticadenocarcinoma cells. However, staining of these cells by the F7 mAb inflow cytometry experiments showed minimal in vitro expression. Althoughthe tumor cells in vivo were stainable by the mAb byimmunohistochemistry, it is unclear at what stage of their tumor growthCXCR4 began to be expressed. This suggests that expression of CXCR4 bythese two cell lines was insufficient to allow for tumor growthinhibition or induction of apoptosis in vivo by anti-CXCR4 treatment.

Example 13 Inhibition of Lung Metastases In Vivo by Anti-CXCR4Antibodies

In this example, the ability of the F7 anti-CXCR4 mAb to inhibit lungmetastases was examined using a C57 mouse systemic tumor model. Morespecifically, 0.4×10⁶ B16-CXCR4 cells (B16 cells transfected to expresshuman CXCR4) were injected intravenously into each of 30 mice of the C57strain. The mice were randomized into three groups of ten mice each,which were then treated with either (i) vehicle (PBS); (ii) IgG4 isotypecontrol (5 mg/kg); or (iii) F7 IgG4 (5 mg/kg). The antibody or vehiclewas injected intraperitoneally 30 minutes after the B16-CXCR4 cells wereinjected intravenously. Lungs were harvested on day 14 and the number oflung metastatic nodules was quantitated. The results are summarizedbelow in Table 5, which shows the mean and median number of lungmetastases in each group:

TABLE 5 Inhbition of Lung Metastases In Vivo by mAb F7 % InhibitionNumber of Lung Metastases of Lung Mets Treatment Mean Median (Mean)Vehicle 364 397 IgG4 Isotype Control 309 294 15% F7 Anti-CXCR4 IgG4 157186 56%The results show that treatment with the F7 mAb led to a reduction inthe mean number of lung metastatic nodules of 56%, whereas reduction wasonly 15% with the isotype control antibody, demonstrating that the F7mAb is capable of inhibiting lung metastases in a systemic tumor model.

What is claimed:
 1. A monoclonal antibody, or an antigen-binding portionthereof, which cross-competes for binding to human CXCR4 with areference antibody or reference antigen-binding portion thereof, whereinthe reference antibody or portion thereof comprises: (a) a heavy chainvariable region comprising amino acids having the sequence set forth inSEQ ID NO: 25 and a light chain variable region comprising amino acidshaving the sequence set forth in SEQ ID NO: 29; (b) a heavy chainvariable region comprising amino acids having the sequence set forth inSEQ ID NO: 26 and a light chain variable region comprising amino acidshaving the sequence set forth in SEQ ID NO: 30; (c) a heavy chainvariable region comprising amino acids having the sequence set forth inSEQ ID NO: 27 and a light chain variable region comprising amino acidshaving the sequence set forth in SEQ ID NO: 31; or (d) a heavy chainvariable region comprising amino acids having the sequence set forth inSEQ ID NO: 28 and a light chain variable region comprising amino acidshaving the sequence set forth in SEQ ID NO: 32; and further wherein themonoclonal antibody or portion thereof comprises a heavy chain variableregion comprising amino acids having a sequence derived from a humanV_(H) 3-48 germline sequence as set forth in SEQ ID NO:49 and a lightchain variable region comprising amino acids having a sequence derivedfrom a human V_(K) L15 germline sequence as set forth in SEQ ID NO:50.2. The monoclonal antibody or antigen-binding portion thereof of claim1, which comprises: (a) a heavy chain variable region CDR1 comprisingamino acids having the sequence set forth in SEQ ID NO: 1 orconservative modifications thereof; (b) a heavy chain variable regionCDR2 comprising amino acids having the sequence set forth in SEQ ID NO:5 or conservative modifications thereof; (c) a heavy chain variableregion CDR3 comprising amino acids having the sequence set forth in SEQID NO: 9; (d) a light chain variable region CDR1 comprising amino acidshaving the sequence set forth in SEQ ID NO: 13 or conservativemodifications thereof; (e) a light chain variable region CDR2 comprisingamino acids having the sequence set forth in SEQ ID NO: 17 orconservative modifications thereof; and (f) a light chain variableregion CDR3 comprising amino acids having the sequence set forth in SEQID NO: 21 or conservative modifications thereof.
 3. The monoclonalantibody or antigen-binding portion thereof of claim 1, which comprises:(a) a heavy chain variable region CDR1 comprising amino acids having thesequence set forth in SEQ ID NO: 2 or conservative modificationsthereof; (b) a heavy chain variable region CDR2 comprising amino acidshaving the sequence set forth in SEQ ID NO: 6 or conservativemodifications thereof; (c) a heavy chain variable region CDR3 comprisingamino acids having the sequence set forth in SEQ ID NO: 10; (d) a lightchain variable region CDR1 comprising amino acids having the sequenceset forth in SEQ ID NO: 14 or conservative modifications thereof; (e) alight chain variable region CDR2 comprising amino acids having thesequence set forth in SEQ ID NO: 18 or conservative modificationsthereof; and (f) a light chain variable region CDR3 comprising aminoacids having the sequence set forth in SEQ ID NO: 22 or conservativemodifications thereof.
 4. The monoclonal antibody or antigen-bindingportion thereof of claim 1, which comprises: (a) a heavy chain variableregion CDR1 comprising amino acids having the sequence set forth in SEQID NO: 3 or conservative modifications thereof; (b) a heavy chainvariable region CDR2 comprising amino acids having the sequence setforth in SEQ ID NO: 7 or conservative modifications thereof; (c) a heavychain variable region CDR3 comprising amino acids having the sequenceset forth in SEQ ID NO: 11; (d) a light chain variable region CDR1comprising amino acids having the sequence set forth in SEQ ID NO: 15 orconservative modifications thereof; (e) a light chain variable regionCDR2 comprising amino acids having the sequence set forth in SEQ ID NO:19 or conservative modifications thereof; and (f) a light chain variableregion CDR3 comprising amino acids having the sequence set forth in SEQID NO: 23 or conservative modifications thereof.
 5. The monoclonalantibody or antigen-binding portion thereof of claim 1, which comprises:(a) a heavy chain variable region CDR1 comprising amino acids having thesequence set forth in SEQ ID NO: 4 or conservative modificationsthereof; (b) a heavy chain variable region CDR2 comprising amino acidshaving the sequence set forth in SEQ ID NO: 8 or conservativemodifications thereof; (c) a heavy chain variable region CDR3 comprisingamino acids having the sequence set forth in SEQ ID NO: 12; (d) a lightchain variable region CDR1 comprising amino acids having the sequenceset forth in SEQ ID NO: 16 or conservative modifications thereof; (e) alight chain variable region CDR2 comprising amino acids having thesequence set forth in SEQ ID NO: 20 or conservative modificationsthereof; and (f) a light chain variable region CDR3 comprising aminoacids having the sequence set forth in SEQ ID NO: 24 or conservativemodifications thereof.
 6. The monoclonal antibody or antigen bindingportion thereof of claim 1 comprising: (a) a heavy chain variable regioncomprising amino acids having a sequence chosen from SEQ ID Nos. 25-28and 41-44 and conservative modifications thereof; and (b) a light chainvariable region comprising amino acids having a sequence chosen from SEQID Nos. 29-32 and 45-48 and conservative modifications thereof.
 7. Themonoclonal antibody or antigen binding portion thereof of claim 6, whichcomprises: (a) a heavy chain variable region comprising amino acidshaving the sequence set forth in SEQ ID NO: 25 or 41 or conservativemodifications thereof; and (b) a light chain variable region comprisingamino acids having the sequence set forth in SEQ ID NO: 29 or 45 orconservative modifications thereof.
 8. The monoclonal antibody orantigen binding portion thereof of claim 6, which comprises: (a) a heavychain variable region comprising amino acids having the sequence setforth in SEQ ID NO: 26 or 42 or conservative modifications thereof; and(b) a light chain variable region comprising amino acids having thesequence set forth in SEQ ID NO: 30 or 46 or conservative modificationsthereof.
 9. The monoclonal antibody or antigen binding portion thereofof claim 6, which comprises: (a) a heavy chain variable regioncomprising amino acids having the sequence set forth in SEQ ID NO: 27 or43 or conservative modifications thereof; and (b) a light chain variableregion comprising amino acids having the sequence set forth in SEQ IDNO: 31 or 47 or conservative modifications thereof.
 10. The monoclonalantibody or antigen binding portion thereof of claim 6, which comprises:(a) a heavy chain variable region comprising amino acids having thesequence set forth in SEQ ID NO: 28 or 44 or conservative modificationsthereof; and (b) a light chain variable region comprising amino acidshaving the sequence set forth in SEQ ID NO: 32 or 48 or conservativemodifications thereof.
 11. The monoclonal antibody or antigen-bindingportion thereof of claim 1, which binds to native human CXCR4 expressedon a cell surface.
 12. The monoclonal antibody or antigen-bindingportion thereof of claim 1, which inhibits binding of SDF-1 to humanCXCR4.
 13. The monoclonal antibody or antigen-binding portion thereof ofclaim 1, which does not inhibit binding of SDF-1 to human CXCR4.
 14. Themonoclonal antibody or antigen-binding portion thereof of claim 1, whichinhibits SDF-1-induced calcium flux in cells expressing human CXCR4. 15.The monoclonal antibody or antigen-binding portion thereof of claim 1,which inhibits SDF-1-induced migration of cells expressing human CXCR4.16. The monoclonal antibody or antigen-binding portion thereof of claim1, which inhibits capillary tube formation by human umbilical veinendothelial cells.
 17. The monoclonal antibody or antigen-bindingportion thereof of claim 1, which induces apoptosis in cells expressinghuman CXCR4.
 18. The monoclonal antibody or antigen-binding portionthereof of claim 1, which inhibits growth or induces apoptosis of CXCR4⁺tumor cells in vivo.
 19. The monoclonal antibody or antigen-bindingportion thereof of claim 1, which binds to human CXCR4 with a K_(D) of1×10⁻⁷ M or less.
 20. The monoclonal antibody or antigen-binding portionthereof of claim 19, which binds to human CXCR4 with a K_(D) of 5×10⁻⁸ Mor less.
 21. The monoclonal antibody or antigen-binding portion thereofof claim 1, which: (a) binds to native human CXCR4 expressed on a cellsurface; (b) inhibits binding of SDF-1 to human CXCR4; (c) inhibitsSDF-1-induced calcium flux in cells expressing human CXCR4; (d) inhibitsSDF-1-induced migration of cells expressing human CXCR4; and (e)inhibits capillary tube formation by human umbilical vein endothelialcells.
 22. The monoclonal antibody or antigen-binding portion thereof ofclaim 21, which induces apoptosis in cells expressing human CXCR4. 23.The monoclonal antibody or antigen-binding portion thereof of claim 21,which inhibits growth or induces apoptosis of CXCR4⁺ tumor cells invivo.
 24. The monoclonal antibody or antigen-binding portion thereof ofclaim 21, which inhibits binding of SDF-1 to human CXCR4 with an EC₅₀ of50 nM or less.
 25. The monoclonal antibody or antigen-binding portionthereof of claim 21, which inhibits SDF-1-induced calcium flux in cellsexpressing human CXCR4 with an EC₅₀ of 3 nM or less.
 26. The monoclonalantibody or antigen-binding portion thereof of claim 21, which inhibitsSDF-1-induced migration of cells expressing human CXCR4 with an EC₅₀ of50 nM or less.
 27. A composition comprising the monoclonal antibody orantigen-binding portion thereof of any of claims 1, 2 and 3 and apharmaceutically acceptable carrier.
 28. An immunoconjugate comprisingthe monoclonal antibody or antigen-binding portion thereof of claim 1 or2 linked to a therapeutic agent.
 29. The immunoconjugate of claim 28,wherein the therapeutic agent is a cytotoxin or a radioactive isotope.30. A composition comprising the immunoconjugate of claim 28 and apharmaceutically acceptable carrier.
 31. A bispecific moleculecomprising the monoclonal antibody or antigen-binding portion thereof ofclaim 1 or 2 linked to a second functional moiety having a differentbinding specificity than said monoclonal antibody or antigen-bindingportion thereof.
 32. A composition comprising the bispecific molecule ofclaim 31 and a pharmaceutically acceptable carrier.
 33. The monoclonalantibody or antigen-binding portion thereof of claim 1, which is an IgG1or IgG4 antibody or a portion thereof.
 34. The antigen-binding portionof the human monoclonal antibody of claim 1, which is a Fab, Fab′ orF(ab′)₂, Fv, dAb, or scFv fragment.
 35. A human monoclonal antibody, oran antigen-binding portion thereof, which specifically binds to nativehuman CXCR4 expressed on a cell surface and comprises a heavy chainvariable region CDR1 comprising amino acids having the sequence setforth in SEQ ID NO: 1; a heavy chain variable region CDR2 comprisingamino acids having the sequence set forth in SEQ ID NO: 5; a heavy chainvariable region CDR3 comprising amino acids having the sequence setforth in SEQ ID NO: 9; a light chain variable region CDR1 comprisingamino acids having the sequence set forth in SEQ ID NO: 13; a lightchain variable region CDR2 comprising amino acids having the sequenceset forth in SEQ ID NO: 17; and a light chain variable region CDR3comprising amino acids having the sequence set forth in SEQ ID NO: 21.36. A human monoclonal antibody, or an antigen-binding portion thereof,which specifically binds to native human CXCR4 expressed on a cellsurface and comprises a heavy chain variable region CDR1 comprisingamino acids having the sequence set forth in SEQ ID NO: 2; a heavy chainvariable region CDR2 comprising amino acids having the sequence setforth in SEQ ID NO: 6; a heavy chain variable region CDR3 comprisingamino acids having the sequence set forth in SEQ ID NO: 10; a lightchain variable region CDR1 comprising amino acids having the sequenceset forth in SEQ ID NO: 14; a light chain variable region CDR2comprising amino acids having the sequence set forth in SEQ ID NO: 18;and a light chain variable region CDR3 comprising amino acids having thesequence set forth in SEQ ID NO:
 22. 37. The monoclonal antibody orantigen binding portion thereof of claim 35, which comprises: (a) aheavy chain variable region comprising amino acids having the sequenceset forth in SEQ ID NO: 25; and (b) a light chain variable regioncomprising amino acids having the sequence set forth in SEQ ID NO: 29.38. The monoclonal antibody or antigen binding portion thereof of claim36, which comprises: (a) a heavy chain variable region comprising aminoacids having the sequence set forth in SEQ ID NO: 26; and (b) a lightchain variable region comprising amino acids having the sequence setforth in SEQ ID NO:
 30. 39. The monoclonal antibody or antigen-bindingportion thereof of any of claims 35-38, which is an IgG1 or IgG4antibody or a portion thereof
 40. The monoclonal antibody orantigen-binding portion thereof of claim 35 or 37, which is an IgG4antibody or a portion thereof.
 41. The monoclonal antibody orantigen-binding portion thereof of claim 36 or 38, which is an IgG1antibody or a portion thereof.
 42. The monoclonal antibody orantigen-binding portion thereof of claim 1, which is a human antibody ora portion thereof.